Means and methods for diagnosing and treating affective disorders

ABSTRACT

The present invention relates to nucleic acid molecules, preferably genomic sequences, encoding an ATP-gated ion channel P2X7R which contain a mutation in the 5′UTR or 3′UTR regions, a mutation in exon 3, 5, 6, 8 or 13 or in introns 1, 3, 4, 5, 6, 7, 9, 11 or 12 or a deletion in exon 13, which allow to diagnose affective disorders. The invention further relates to polypeptides encoded by said nucleic acid molecules vectors and host cells comprising said nucleic acid molecules as well as to methods for producing polypeptides encoded by said nucleic acid molecules. The present invention also provides antibodies specifically directed to polypeptides encoded by said nucleic acid molecules and aptamers specifically binding said nucleic acid molecules. Additionally, primers for selectively amplifying said nucleic acid molecules are provided in the present invention as well as kits, compositions, particularly pharmaceutical and diagnostic compositions comprising said nucleic acid molecules, vectors, polypeptides, aptamers, antibodies and/or primers. Moreover, the present invention relates to methods for diagnosing affective disorders associated with a nonfunctional P2X7R protein, an altered ATP-gating of the P2X7R protein, an over- or underexpression of the P2X7R protein or associated with the presence of any one of the aforementioned nucleic acid molecules or polypeptides encoded thereby. Additionally, the present invention relates to uses and methods for treating affective disorders employing a functional or non-functional ATP-gated ion-channel P2X7R. The present invention also relates to uses of modulators of P2X7R activity for treating affective diseases. Furthermore, the present invention also relates to methods for identifying and characterizing compounds which are capable of specifically interacting with or altering the characteristics of the polypeptides of the present invention as well as to methods for the production of pharmaceutical compositions.

The present invention relates to nucleic acid molecules, preferablygenomic sequences, encoding an ATP-gated ion channel P2X7R which containa mutation in the 5′UTR or 3′UTR regions, a mutation in exon 3, 5, 6, 8or 13 or in introns 1, 3, 4, 5, 6, 7, 9, 11 or 12 or a deletion in exon13, which allow to diagnose affective disorders. The invention furtherrelates to polypeptides encoded by said nucleic acid molecules vectorsand host cells comprising said nucleic acid molecules as well as tomethods for producing polypeptides encoded by said nucleic acidmolecules. The present invention also provides antibodies specificallydirected to polypeptides encoded by said nucleic acid molecules andaptamers specifically binding said nucleic acid molecules.

Additionally, primers for selectively amplifying said nucleic acidmolecules are provided in the present invention as well as kits,compositions, particularly pharmaceutical and diagnostic compositionscomprising said nucleic acid molecules, vectors, polypeptides, aptamers,antibodies and/or primers. Moreover, the present invention relates tomethods for diagnosing affective disorders associated with anon-functional P2X7R protein, an altered ATP-gating of the P2X7Rprotein, an over- or underexpression of the P2X7R protein or associatedwith the presence of any one of the aforementioned nucleic acidmolecules or polypeptides encoded thereby. Additionally, the presentinvention relates to uses and methods for treating affective disordersemploying a functional or non-functional ATP-gated ion-channel P2X7R.The present invention also relates to uses of modulators of P2X7Ractivity for treating affective diseases.

Furthermore, the present invention also relates to methods foridentifying and characterizing compounds which are capable ofspecifically interacting with or altering the characteristics of thepolypeptides of the present invention as well as to methods for theproduction of pharmaceutical compositions.

Up to 10% of persons visiting a physician are afflicted with anaffective disorder (also known as behavioural disorder, mood disorder).Nonetheless, most cases remain undiagnosed or inadequately treated.Affective disorders include among others, depression, anxiety, andbipolar disorder. These diseases are well described in the literature;see, for example, Diagnostic and Statistical Manual of MentalDisorders-4th Edition Text Revision (DMS-IV-TR), American PsychiatricPress, 2000.

Depression, also known as unipolar affective disorder, is characterizedby a combination of symptoms such as lowered mood, loss of energy, lossof interest, feeling of physical illness, poor concentration, alteredappetite, altered sleep and a slowing down of physical and mentalfunctions resulting in a relentless feeling of hopelessness,helplessness, guilt, and anxiety. The primary subtypes of this diseaseare major depression, dysthymia (milder depression), and atypicaldepression. Other important forms of depression are premenstrualdysphoric disorder and seasonal affective disorder. Present treatment ofdepression consists of psychotherapy, antidepressant drugs, or acombination of both. Most antidepressive drugs target the transport ofthe neurotransmitters serotonin and/or norepinephrine, or the activityof the enzyme monoamine oxidase. They include: Selectiveserotonin-reuptake inhibitors (e.g., fluoxetine, paroxetine, sertraline,fluvoxamine), tricyclic antidepressants (e.g., amitriptyline,imipramine, desipramine, nortriptyline), monoamine oxidase inhibitors(e.g., phenelzine, isocarboxazid, tranylcypromine), and designerantidepressants such as mirtazapine, reboxetine, nefazodone. However,all existing antidepressive drugs possess shortcomings such as longlatency until response, high degree of non-responders and undesirableside effects (Holsboer, Biol. Psychol. 57 (2001), 47-65). Therefore, aneed exists in the medical community for new antidepressive drugs withimproved pharmacological profile (Baldwin, Hum. Psychopharmacol. Clin.Exp. 16 (2001), S93-S99).

Anxiety disorders are defined by an excessive or inappropriate arousedstate characterized by feelings of apprehension, uncertainty, or fear.They are classified according to the severity and duration of theirsymptoms and specific affective characteristics. Categories include: (1)Generalized anxiety disorder, (2) panic disorder, (3) phobias, (4)obsessive-compulsive disorder, (5) post-traumatic stress disorder, and(6) separation anxiety disorder. The standard treatment for most anxietydisorders is a combination of cognitive-behavioural therapy withantidepressant medication. Additional medications includebenzodiazepines and buspirone.

Bipolar disorder, also known as manic-depression, is characterized bymood swings between periods of mania (i.e. mood elevation includingexaggerated euphoria, irritability) and periods of depression. Bipolardisorder is classified according to the severity of the symptoms.Patients diagnosed with bipolar disorder type I suffer from manic ormixed episodes with or without major depression. In Bipolar Disordertype II, patients have episodes of hypomania and episodes of majordepression. With hypomania the symptoms of mania (euphoria orirritability) appear in milder forms and are of shorter duration. Thecurrent drugs used to treat bipolar disorders are lithium, valproate andlamotrigine, which stimulates the release of the neurotransmitterglutamate. As with antidepressive drugs, they take weeks to becomeeffective and can result in undesirable side effects, for example, highlevels of lithium in the blood can be fatal.

Compelling evidence suggest that affective disorders are biologicaldiseases. However, there are no laboratory tests or other proceduresthat a common physician can use to make a definitive diagnosis. Instead,a specially trained physician or psychiatrist must diagnose the illnessbased on a group of symptoms that occur together. This process is oftentime consuming and laborious requiring several visits for the physicianto perform a careful history of the symptoms that the patient iscurrently experiencing as well as any symptoms he or she has had in thepast. Therefore, an easy and effective method for the accurate diagnosisof affective disorders is of high interest to the medical community(Wittchen et al., J. Clin. Psychiatry 62, suppl. 26 (2001), 23-28).

Most patients afflicted with affective disorders have family antecedentsand identical twins studies suggest a strong genetic component. Forexample, genetic mapping on an isolated population of the central valleyof Costa Rica suggests a locus for severe bipolar disorder at chromosome18q22-q23 (Freimer et al., Nature Genetics 12 (1996), 436-441).Moreover, genetic studies performed on the Old Order Amish populationsuggest that genes on chromosomes 6, 13, and 15 may contribute to thesusceptibility of bipolar affective disorder (Ginns et al., NatureGenetics 12 (1996), 431-435). Recently, a genome-wide search in ahomogenous population found in the Saguenay/Lac-St-Jean region of Quebecsuggests the presence of a major locus for bipolar disorder onchromosome 12q23-q24 (Morissette et al., Am. J. Med. Genet.(Neuropsychiatr. Genet.) 88 (1999), 567-587). Susceptibility loci onchromosomes 5 and 21 were also found in this study. Other groups reportminimal evidence for linkage in the region of 12q23 (Kelsoe et al.,Proc. Natl. Acad. Sci. USA 98 (2001), 585-590; Sklar, Annu. Rev.Genomics Hum. Genet. 3 (2002), 371-413). Given the various locimentioned in the above studies (e.g., links to chromosomes 5, 6, 12, 13,15, 18, 21), a definite genetic link for affective diseases remains tobe found.

Thus, although several genes have been assumed to be linked withaffective disorders as mentioned hereinabove, however, no clearcorrelation has so far been shown. Since no well-suited medication nordiagnosis on a molecular level for affective disorders is available,there is a need for identifying a gene whose mutations cause the wholespectrum of affective disorders as well as for providing medicaments andmethods for diagnosis and treatment of affective disorders.

Thus, the technical problem underlying the present invention is toprovide means and methods for diagnosis and treating affectivedisorders.

The solution to said technical problem is achieved by providing theembodiments characterized in the claims.

Accordingly, the present invention relates to a nucleic acid moleculecomprising a nucleic acid sequence selected from the group consistingof:

-   (a) a genomic nucleotide sequence encoding an ATP-gated ion channel    P2X7R and which contains a mutation in the 5′UTR region    corresponding to positions 362, 532, 1100, 1122, 1171 or 1702 of the    genomic sequence of the wild-type ATP-gated ion channel P2X7R as    depicted in SEQ ID NO: 1, wherein at said position said nucleotide    is replaced by another nucleotide;

(b) a nucleic acid sequence encoding a polypeptide which has an aminoacid sequence of the ATP-gated ion channel P2X7R, wherein in the exon asindicated in column “Exon” of the following Table A the amino acidresidue as indicated in column “Amino acid residue” of Table Acorresponding to the position as indicated in column “Position inwild-type” of Table A of the wild-type ATP-gated ion channel P2X7R aminoacid sequence as depicted in SEQ ID NO: 3 or 4 is replaced by anotheramino acid residue TABLE A Exon Amino acid residue Position in wild-typeexon 3 R (Arg) 117 exon 5 G (Gly) 150 exon 6 E (Glu) 186 exon 6 L (Leu)191 exon 8 R (Arg) 270 exon 13 I (Ile) 568 exon 13 R (Arg) 578

-   (c) a nucleotide sequence encoding an ATP-gated ion channel P2X7R    and which contains a mutation in exon 5 or 8 corresponding to    position 32548 or position 37633 of the wild-type ATP-gated ion    channel P2X7R nucleotide sequence as depicted in SEQ ID NO: 1,    wherein at said position said nucleotide is replaced by another    nucleotide-   (d) a nucleic acid sequence encoding a polypeptide which has an    amino acid sequence of an ATP-gated ion channel P2X7R, wherein amino    acids corresponding to positions 488 to 494 of the wild-type    ATP-gated ion channel P2X7R as depicted in SEQ ID NO: 3 or 4 are    deleted;

(e) a genomic nucleotide sequence encoding an ATP-gated ion channelP2X7R, wherein in the intron as indicated in column “Intron” of thefollowing Table B the nucleotide as indicated in column “Replacednucleotide” of Table B corresponding to the position as indicated incolumn “Position in wild-type” of Table B of the wild-type ATP-gated ionchannel P2X7R nucleotide sequence as depicted in SEQ ID NO: 1 isreplaced by another nucleotide TABLE B Intron REPLACED NUCLEOTIDEPosition in wild-type intron 1 G 3166 intron 1 C 24778 intron 1 C 24830intron 3 A 26308 intron 3 G 26422 intron 4 G 32394 intron 4 T 32434intron 5 A 32783 intron 6 G 35641 intron 6 A 35725 intron 6 T 36001intron 7 G 36378 intron 7 T 36387 intron 7 G 36398 intron 9 C 47214intron 11 T 47563 intron 12 C 54307 intron 12 G 54308

-   (f) a genomic nucleotide sequence encoding an ATP-gated ion channel    P2X7R and which contains a mutation in the 3′UTR region    corresponding to position 55169, 55170, 55171, 55917 or 54925 of the    wild-type ATP-gated ion channel P2X7R nucleotide sequence as    depicted in SEQ ID NO: 1, wherein at said position said nucleotide    is replaced by another nucleotide;-   (g) a nucleotide sequence comprising at least 20 or 21 nucleotides    and comprising the mutations or deletions as defined in any one    of (a) to (f);-   (h) a nucleic acid sequence comprising a nucleotide sequence as    shown in any one of SEQ ID NOs: 13 to 51;-   (i) a nucleic acid sequence encoding a polypeptide comprising the    amino acid sequence of SEQ ID NOs: 5 to 12;-   (j) a nucleotide sequence which hybridizes to a nucleotide sequence    defined in any one of (a) to (g) or to the nucleotide sequence    of (h) and having a mutation as defined in any one of (a) to (f);    and-   (k) a nucleic acid sequence being degenerate as a result of the    genetic code to the nucleic acid sequence as defined in (j).

It has surprisingly been found that mutations in the P2X7R gene whichencodes the ATP-gated ion channel P2X7R can cause the whole spectrum ofaffective disorders. Six different mutations in the 5′UTR of the P2X7Rgene, seven different mutations in exons 3, 5, 6, 8 and 13 of the P2X7Rgene leading to an amino acid replacement of the corresponding aminoacid in the wild-type sequence of P2X7R depicted in SEQ ID NO: 3 or 4and two mutations in exons 5 and 8 of said gene, respectively, leadingto a replacement of a nucleotide by another nucleotide, a deletion ofnucleotides in exon 13 of said gene, 18 mutations in introns 1, 3, 4, 5,6, 7, 9, 11 and 12 and 5 mutations in the 3′UTR of the P2X7R gene havebeen identified to co-segregate with the affection status in 41unrelated families affected with affective disorders. The term“affective disorder” when used in the context of the present inventionmeans to include, but is not limited to, depression, anxiety, unipolardisorder, bipolar disorder type I, bipolar disorder type II, mania,attention deficit hyperactive disorder, substance abuse, and any otherdisorders affecting the normal behaviour, or mood of an individual.

Each mutation causes alterations that can explain affective disorders asshown in the Examples hereinbelow.

P2X7R is an ATP-gated ion channel belonging to the P2X ionotropicchannel family. The gene was first isolated from rat brain (Surprenantet al., (1996), 272, 735-738; Genbank accession number NM_(—)019256) andsubsequently from a human monocyte library (Rassendren et al., J. Biol.Chem. 272 (1997), 5482-5486; Genbank accession numbers NM_(—)002562,Y09561) by virtue of its sequence homology with the other members of theP2X family. It was later found that P2X7R corresponded to theunidentified P2Z receptor which mediates the permeabilising action ofATP on mast cells and macrophages (Dahlqvist and Diamant, Acta Physiol.Scand. 34 (1974), 368-384; Steinberg and Silverstein, J. Biol. Chem. 262(1987), 3118-3122; Gordon, Biochem. J. 233 (1986), 309-319). The P2X7Rhas two hydrophobic membrane-spanning domains, an extracellular loop,and forms transmembrane ion channels. P2X7 receptors seem to functiononly in homooligomeric form and bear a pharmacological profile markedlydifferent from other P2X homo- or heteromers (North and Surprenant,Annual Rev. Pharmacology Toxicology 40 (2000), 563-580). P2X7R requireslevels of ATP in excess of 1 mM to achieve activation, whereas other P2Xreceptors activate at ATP concentrations of ≦100 μM (Steinberg et al.,J. Biol. Chem. 262 (1987), 8884-8888; Greenberg et al., J. Biol. Chem.263 (1988), 10337-10343) 32). While all P2X receptors demonstratenon-selective channel-like properties following ligation, the channelsformed by the P2X7R can rapidly transform into pores that can allow thepassage of molecules of up to 900 Dalton (Virainio et al., J. Physiol.519 (1999), 335-346).

P2X7R is expressed in hematopoietic cells, mast cells and macrophages(Surprenant et al., Science 272 (1996), 3118-3122), where it isorganized in tetrameric or hexameric form (Kim et al., J. Biol. Chem.276 (2001), 23262-23267). P2X7R is inter alia involved in the regulationof the immune function and inflammatory response.

Activation of P2X7R by ATP in macrophages is associated with mitogenicstimulation of T cells (Baricordi et al., Blood 87 (1996), 682-690), therelease of cytokines such as interleukin-1β (Griffiths et al., J. Immol.154 (1995), 2821-2828), and formation of macrophage polykarions (Falzoniet al., J. Clin. Invest. 95 (1995), 1207-1216). Stimulation of the P2X7Rwith ATP can also result in cell death by triggering massivetransmembrane ion fluxes (particularly influx of Ca2+ and Na+, andefflux of K+) and the formation of non-selective plasma membrane pores(Di Virgilio et al., Cell Death Differ. 5 (1998), 191-199).

In the brain, P2X7R was originally thought to be restricted to microglia(resident macrophage of the brain) and ependymal cells rather thanneurons (Collo et al., Neuropharmacology 36 (1997), 1277-1283)suggesting a role of P2X7R in neurodegeneration. However, P2X7R hassince been found in neurons of the rat retina (Brandle et al, BrainResearch Molecular Brain Res. 62 (1998), 106-109), cochlear ganglioncells (Brandle et al, Neuroscience Letters 273 (1999), 105-108), andpresynaptic terminals of neurons throughout the brainstem and spinalcord (Deuchards et al., J. Neurosci. 21 (2001), 7143-7152). Subsequentstudies also suggest that P2X7R regulates the release ofneurotransmitters such as glutamate and GABA in neurons of thehippocampus (Armstrong et al., J. Neuroscience 22 (2002), 5938-5945,Sperlágh et al., J. Neurochem. 81 (2002), 1196-1211). Organisation ofP2X7R in glial cells and astrocytes of the brain appears monomeric (Kimet al., J. Biol. Chem. 276 (2001), 23262-23267).

Several agonists and antagonists of P2X7R have been identified.Brilliant Blue (Jiang et al., Mol. Phamacol. 58 (2000), 82-88), theisoquinolines1-[N,O-Bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazineand N-[1-[N-methyl-p-(5 isoquinolinesulfonyl) benzyl]-2-(4phenylpiperazine)ethyl]-5-isoquinolinesulfonamide (Humphreys et al.,Mol. Pharmacol., 54 (1998), 22-32), adamantane derivatives (WO 99/29660,WO 99/29661, WO 00/61569, WO 01/42194, WO 01/44170, WO 01/44213),substituted phenyl compounds (WO 00/71529), piperidine and piperazinederivatives (WO 01/46200) are antagonists of P2X7R while Oxidized ATP(oATP) acts as an irreversible inhibitor of the receptor (Chen et al.,J. Biol. Chem., 268 (1993), 8199-8203). Some of these antagonists arepresently being evaluated for the treatment of inflammatory, immune, andcardiovascular diseases. BzATP (2′-3′-O-(4-Benzoylbenzoyl)adenosine5′-triphosphate (C₂₄H₂₄N₅O₁₅P₃)) acts as agonist of P2X7R (North andSurprenant, Annu. Rev. Pharmacol. Toxicol. 40 (2000), 563-580). WO99/55901 describes a method for identifying compounds that modulate theactivity of a mammalian purinoreceptor selected from the groupconsisting of P2X2, P2X3, P2X4, P2X5, P2X6 and P2)(7 and suggests a roleof said purinoreceptors in therapy of behavioural disorders such asepilepsy, depression and aging-associated degenerative diseases.

Mutant mice lacking P2X7R are healthy, fertile and demonstrate no overtphenotype. However, in contrast to their wild-type counterparts,LPS-activated peritoneal macrophages from P2X7^(−/−) animals fail togenerate mature interleukin-1β (IL-1β) when challenged with ATPsuggesting an inability of peritoneal macrophages to release IL-1 inresponse to ATP (Solle et al, J. Biol. Chem. 276 (2001), 125-132). Adetailed behavioural study of the P2X7R−/− mice was not performed. Inhumans, a Glu-496 to Ala polymorphism leads to the loss of P2X7 function(u et al., J. Biol. Chem. 276 (2001), 11135-11142) and is associatedwith B-cell chronic lymphocytic leukaemia (Thunberg, et al, The Lancet360 (2002), 1935-1939). Additional polymorphs in the putative P2X7Rpromoter region, and coding region have been reported (Li et al., FEBSLett. 531 (2002), 127-131; EP 1199372).

Despite the abundant literature concerning P2X7R, a role in affectivedisorders has never been suggested or alluded to in the prior art.

Before the present invention is described in detail, it is to beunderstood that this invention is not limited to the particularmethodology, protocols, cell lines, vectors, and reagents describedherein as these may vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to limit the scope of the presentinvention which will be limited only by the appended claims. Unlessdefined otherwise, all technical and scientific terms used herein havethe same meanings as commonly understood by one of ordinary skill in theart.

Preferably, the terms used herein are defined as described in “Amultilingual glossary of biotechnological terms: (IUPACRecommendations)”, Leuenberger, H. G. W, Nagel, B. and Kölbl, H. eds.(1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland).

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the word “comprise”, and variations such as“comprises” and “comprising”, will be understood to imply the inclusionof a stated integer or step or group of integers or steps but not theexclusion of any other integer or step or group of integer or step.

Several documents are cited throughout the text of this specification.Each of the documents cited herein (including all patents, patentapplications, scientific publications, manufacturers specifications,instructions, etc.), whether supra or infra, are hereby incorporated byreference in their entirety. Nothing herein is to be construed as anadmission that the invention is not entitled to antedate such disclosureby virtue of prior invention.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the”, include plural referents unless thecontext clearly indicates otherwise. Thus, for example, reference to “areagent” includes one or more of such different reagents, and referenceto “the method” includes reference to equivalent steps and methods knownto those of ordinary skill in the art that could be modified orsubstituted for the methods described herein.

In accordance with the present invention, the term “nucleic acidsequence” means the sequence of bases comprising purine- and pyrimidinebases which are comprised by nucleic acid molecules, whereby said basesrepresent the primary structure of a nucleic acid molecule. Nucleic acidsequences include DNA, cDNA, genomic DNA, RNA, synthetic forms and mixedpolymers, both sense and antisense strands, or may contain non-naturalor derivatized nucleotide bases, as will be readily appreciated by thoseskilled in the art.

When used herein, the term “polypeptide” means a peptide, a protein, ora polypeptide which encompasses amino acid chains of a given length,wherein the amino acid residues are linked by covalent peptide bonds.However, peptidomimetics of such proteins/polypeptides wherein aminoacid(s) and/or peptide bond(s) have been replaced by functional analogsare also encompassed by the invention as well as other than the 20gene-encoded amino acids, such as selenocysteine. Peptides,oligopeptides and proteins may be termed polypeptides. The termspolypeptide and protein are often used interchangeably herein. The termpolypeptide also refers to, and does not exclude, modifications of thepolypeptide, e.g., glycosylation, acetylation, phosphorylation and thelike. Such modifications are well described in basic texts and in moredetailed monographs, as well as in a voluminous research literature.

The term “position” used in accordance with the present invention meansthe position of either an amino acid within an amino acid sequencedepicted herein or the position of a nucleotide within a nucleic acidsequence depicted herein.

The term “ATP-gated ion channel P2X7R”, in accordance with thisinvention, denotes a polypeptide which can be classified as a member ofthe P2X ionotropic receptor family. They are also known as purinergicreceptors. P2X receptors are ligand-gated ion channels. The ligand forthese receptors may be ATP and/or another natural nucleotide such asADP, UTP and UDP, or a synthetic nucleotide such as 2-methylthioATP. Thecriteria for the classification are: (1) a sequence homology that ishigher than 39% across the family or different species; (2) signaltransduction mechanism involving ion conductance (Khakh et al.,Pharmacol Rev. 253 (2001), 107-18). Accordingly, the term “ATP-gated ionchannel P2X7R” is interchangeable with the terms “ionotropic receptor”or “purinergic receptor”. Preferably, the term “ATP-gated ion channelP2X7R” denotes a polypeptide which can be classified as an ATP-gated ionchannel P2X7R on the basis of one or more structural and/or functionalcharacteristics, preferably those described above. Structuralcharacteristics refer to certain structural features which allow toclassify a polypeptide as being a P2X7R protein. One such feature is theamino acid sequence. In the context of the present invention apolypeptide is classified as an ATP-gated ion channel P2X7R if it showsa certain degree of sequence identity over its own length to the aminoacid sequence of the human P2X7R protein depicted in SEQ ID NO: 3 or 4.This degree of sequence identity is at least 40%, more preferably atleast 50%, even more preferably at least 60%, at least 70%, at least80%, at least 90% or at least 95%. It is particularly preferred that thedegree of sequence identity is at least 65%.

Moreover, structural characteristics of P2X7R proteins are twohydrophobic membrane-spanning domains, an extra cellular loop whichcould be analyzed by using the program TMPRED (Hofmann Biol. Chem. 347(1993), 166) or TMHMM (Krogh J. Mol. Bio. 305 (2001), 567-580).Additionally, P2X7R may exist as a single polypeptide, as dimer,tetramer or the like.

Thus, in the context of the present invention a protein is preferablyclassified as a P2X7R protein if it displays at least one of theabove-mentioned structural characteristics. Functional characteristicsrefer to properties related to the biological activity of the P2X7Rprotein. In particular, P2X7R is an ATP-gated ion channel which allowscalcium and sodium ions to pass from extracellular solution tointracellular solution, and allows potassium ions to pass fromintracellular to extracellular solution. Moreover, the ATP-gated ionchannel P2X7R forms naturally a homooligomeric form. The characteristicsof P2X7R receptor proteins can be determined as mentioned hereinbelow.The term “ATP-gated ion channels P2X7R” comprises functional andnon-functional forms of the ATP-gated ion channels P2X7R. A functionalATP-gated ion channel P2X7R is understood to be a P2X7R protein whichhas at least one of the above-mentioned functional characteristics whichcan be measured by methods known in the art. A non-functional ATP-gatedion channel P2X7R is a protein which can be classified as a P2X7Rprotein due to structural characteristics as described above but whichhas lost at least one, preferably all, functional characteristics of aP2X7R protein as described above. Non-functionality of the P2X7R proteincan, e.g., be determined by measuring whether calcium and sodium ionscan flow into cells or whether potassium ions can exit from cells. Thus,it is possible to determine the occurrence of a mutation in theATP-gated ion channel P2X7R by measuring either calcium and/or sodiuminflux or efflux of cells. Cells harbouring a mutation in the P2X7R geneshow an altered ion influx and/or efflux in comparison to cellsharbouring a wild-type P2X7R protein.

Additionally, there are different methods that could be used todetermine whether the P2X7R is functional or non-functional, forexample, altered. One method consists of measuring the rate ofATP-induced incorporation of ethidium into cells, e.g. cells isolatedfrom an individual. Ethidium is incorporated into the cells throughP2X7R pores, when the pore formation is activated by ATP. Cells are thenincubated with or without ATP in the presence of ethidium, then they areanalyzed by flow cytometry. Ethidium fluorescence is measured andcompared in the presence or absence of ATP. If the P2X7R has loweractivity, the ethidium fluorescence induced by ATP will be lower than incontrol cells. Such a method was used to verify P2X7R activity inisolated B-lymphocytes and T-lymphocytes from leukaemia patients (Wileyet al., Lancet 359 (2002), 1114-1119). Briefly, isolated cells areincubated in 1 ml of Hepes buffered potassium chloride at 37° C. withcontinuous stirring. Ethidium is then added at a concentration of 25mol/l, followed 40 seconds later by the addition of 10 μl of 100 mmol/lATP stock. Cells are analyzed at 1,000 events/s by flow cytometry usinga Coulter Elite flow cytometer (Coulter, Hialeah, Fla.) with argon laserexcitation at 488 nm. Fluorescent emission was collected using a 590-nmlong-pass filter. The linear mean channel fluorescence intensity foreach gated subpopulation over successive 5-s intervals was analyzed withthe use of Win-MDI software (Joseph Trotter, version 2.7) and plottedagainst time.

Another method of determining P2X7R activity is to measure calcium entryinto isolated cells incubated with fluorescent dyes that emit only uponbinding to calcium. The cells have to be loaded with the dye and thenthe calcium entry has to be stimulated. Examples of such dyes includeFura-2, Calcium green, calcium orange, calcium crimson (all availablefrom Molecular Probes). Methods of measuring calcium transport are wellknown in the art; see for example, Takahashi et al., Physiol Rev. 79(1999), 1089-1125. Furthermore, calcium entry into the cells produceschanges in the membrane electric potential. This changes can be measuredby electrophysiology (patch clamp) or by using dyes which are sensitiveto voltage change. Such methods are also well known in the art, see forexample, Gonzalez et al., DDT 4 (1999), 431-439; González and Tsien,Chemistry & Biology 4 (1997), 269-277; González and Tsien, BiophysicalJournal 69 (1995), 1272-1280.

Yet another method is to measure uptake of 133Ba21. Ba21 is a goodsurrogate for Ca21 and once inside the cell is neither pumped norsequestered by transport ATPases. Ba21 uptake can be measured over 60 susing 133BaCl2 (final concentration, 0.2 mM). At time 0, a prewarmedstock solution of 133Ba21 (0.4 mM and 1 μCi/ml) is added in equalvolumes to prewarmed isolated cells in 150 mM KCl with HEPES (pH 7.4) at37° C. ATP (1 mM) is added either 10 minutes before or simultaneouslywith the 133Ba21 isotope. Aliquots of 0.8 ml are taken at time pointsbetween 0 and 60 s and are immediately mixed with 0.2 ml of ice-cold 50mM MgCl2 (in KCl-HEPES medium) that had been previously layered over 250μl of oil mixture (di-n-butyl phthalate and di-iso-octyl phthalate, 7:3vol/vol) and then centrifuged at 8,000 g for 30 s. The supernatants andthe oil are aspirated, and the cell pellets are counted in a WallacWizard 3 automatic gamma-counter or in any other suitable gammameasuring unit.

The present invention is based on the finding that mutations ofdifferent kinds in the P2X7R gene are linked to the occurrence ofaffective disorders. The first type of mutations are mutations in the5′UTR. Examples of such mutations are single nucleotide replacements atpositions corresponding to positions 362, 532, 1100, 1122, 1171 or 1702of the genomic sequence of the wild-type ATP-gated ion channel P2X7R asdescribed in SEQ ID NO: 1.

The position with respect to nucleotide sequences mentioned herein referto the sequence shown in SEQ ID NO: 1. This sequence represents thenucleic acid sequence of the P2X7R gene encoding the ATP-gated ionchannel P2X7R. It is possible for the skilled person to identify theposition in the genomic sequence corresponding to a position in SEQ IDNO: 1 by aligning the sequences. Moreover, the exact locations of theexons and introns are indicated in SEQ ID NO: 1 hereinbelow.Additionally, the person skilled in the art is able to identify exonsand introns of the P2X7R gene by comparing SEQ ID NO: 1 with SEQ ID NO:2 which shows the cDNA sequence of the P2X7R gene.

Preferably, at position 362 in the 5′UTR of the genomic sequence of theP2X7R gene depicted in SEQ ID NO: 1 a thymine (T) is replaced by anothernucleotide, preferably a purine base. More preferably, at said positionsaid thymidine is replaced by a pyrimidine base. Particularly preferred,said thymine is replaced by a cytosine (C).

At position 532 in the 5′UTR of the genomic sequence of the P2X7R genedepicted in SEQ ID NO: 1 a thymine (T) is preferably replaced by anothernucleotide, preferably a pyrimidine base. More preferably, at saidposition said thymine is replaced by a purine base. Particularlypreferred, said thymidine is replaced by a guanine (G).

The adenine (A) residues at positions 1100 and 1122, respectively, inthe 5′UTR of the genomic sequence of the P2X7R gene is preferablyreplaced by a pyrimidine base. More preferably, said adenine is replacedby a purine base and particularly preferred said adenine is replaced bya guanine (G).

At position 1171 in the 5′UTR of the genomic sequence of the P2X7R genedepicted in SEQ ID NO: 1 a cytidine (C) is replaced by anothernucleotide, preferably by a pyrimidine base. More preferably, saidcytidine is replaced by a purine base and even more preferred, saidcytidine is replaced by a guanine (G).

The guanine at position 1702 in the 5′UTR of the genomic sequence of thegene P2X7R depicted in SEQ ID NO: 1 is replaced by another nucleotide,preferably by a pyrimidine base. More preferably, said guanine isreplaced by a purine base and particularly preferred it is replaced byan adenine (A).

A second type of mutation found in the P2X7R gene are mutations in exonswhich lead to amino acid substitutions in the corresponding amino acidsequence. These are the mutations listed under item (b), supra. In thiscontext, the term “an amino acid residue as indicated in column ‘Aminoacid residue’ of Table A corresponding to position X of the wild-typeATP-gated ion channel P2X7R as depicted in column ‘Position inwild-type’” has the following meaning: The amino acid residue inquestion would be located at position X in the sequence of SEQ ID NO: 3or 4 if the sequence in which said amino acid residue occurs is comparedand aligned with the amino acid sequence of SEQ ID NO: 3 or 4. The aminoacid sequence shown in SEQ ID NO: 3 or 4 is the amino acid sequence ofthe human P2X7R gene and is used as a reference sequence in the presentinvention.

In order to determine whether an amino acid residue or nucleotideresidue in a given P2X7R sequence corresponds to a certain position inthe amino acid sequence or nucleotide sequence of SEQ ID NO: 1, 3 or 4,the skilled person can use means and methods well-known in the art,e.g., alignments, either manually or by using computer programs such asthose mentioned further down below in connection with the definition ofthe term “hybridization” and degrees of homology.

For example, BLAST2.0, which stands for Basic Local Alignment SearchTool (Altschul, Nucl. Acids Res. 25 (1997), 3389-3402; Altschul, J. Mol.Evol. 36 (1993), 290-300; Altschul, J. Mol. Biol. 215 (1990), 403410),can be used to search for local sequence alignments. BLAST producesalignments of both nucleotide and amino acid sequences to determinesequence similarity. Because of the local nature of the alignments,BLAST is especially useful in determining exact matches or inidentifying similar sequences. The fundamental unit of BLAST algorithmoutput is the High-scoring Segment Pair (HSP). An HSP consists of twosequence fragments of arbitrary but equal lengths whose alignment islocally maximal and for which the alignment score meets or exceeds athreshold or cutoff score set by the user. The BLAST approach is to lookfor HSPs between a query sequence and a database sequence, to evaluatethe statistical significance of any matches found, and to report onlythose matches which satisfy the user-selected threshold of significance.The parameter E establishes the statistically significant threshold forreporting database sequence matches. E is interpreted as the upper boundof the expected frequency of chance occurrence of an HSP (or set ofHSPs) within the context of the entire database search. Any databasesequence whose match satisfies E is reported in the program output.

Analogous computer techniques using BLAST (Altschul (1997), loc. cit.;Altschul (1993), loc. cit.; Altschul (1990), loc. cit.) are used tosearch for identical or related molecules in nucleotide databases suchas GenBank or EMBL. This analysis is much faster than multiplemembrane-based hybridizations. In addition, the sensitivity of thecomputer search can be modified to determine whether any particularmatch is categorized as exact or similar. The basis of the search is theproduct score which is defined as:$\frac{\%\quad{sequence}\quad{{identity} \times \%}\quad{maximum}\quad{BLAST}{\quad\quad}{score}}{100}$and it takes into account both the degree of similarity between twosequences and the length of the sequence match. For example, with aproduct score of 40, the match will be exact within a 1-2% error; and at70, the match will be exact. Similar molecules are usually identified byselecting those which show product scores between 15 and 40, althoughlower scores may identify related molecules.

As mentioned above, the second group of mutations identified in theP2X7R gene are mutations in the exons of the P2X7R gene which lead toamino acid substitutions. In this respect SEQ ID NO 2 shows the cDNAsequence of the P2X7R gene. In exon 3 at position 117 of thecorresponding wild-type amino acid sequence of P2X7R depicted in SEQ IDNO: 3 or 4 an arginine (R) residue is replaced by another amino acidresidue, preferably by an aliphatic, acidic or basic amino acid residue.More preferably, by an aromatic amino acid residue which is particularlypreferred to be a tryptophane (W). The resulting polypeptide is shown inSEQ ID NO: 5.

In exon 5 at position 150 of the wild-type amino acid sequence of P2X7Rdepicted in SEQ ID NO: 3 or 4 a glycine (G) residue is replaced byanother amino acid residue, preferably by an aliphatic, aromatic oracidic amino acid residue. More preferably, by a basic amino acidresidue and particularly preferred by an arginine (R). The resultingpolypeptide is shown in SEQ ID NO: 6.

At position 186 in exon 6 of the wild-type amino acid sequence of P2X7Rdepicted in SEQ ID NO: 3 or 4 a glutamate residue (E) is replaced byanother amino acid residue, preferably by an aliphatic, aromatic oracidic amino acid residue. More preferably, said glutamate is replacedby a basic amino acid residue which is particularly preferred a lysine(K). The resulting polypeptide is shown in SEQ ID NO: 7.

In exon 6 of the wild-type amino acid sequence of P2X7R depicted in SEQID NO: 3 or 4 at position 191 a leucine residue (L) is replaced byanother amino acid residue. Said amino acid residue is preferably analiphatic, acidic or basic amino acid residue. More preferably, saidamino acid residue is an aromatic amino acid residue which isparticularly preferred to be a proline (P). The resulting polypeptide isshown in SEQ ID NO: 8.

In exon 8 of the wild-type amino acid sequence of P2X7R depicted in SEQID NO: 3 or 4 at position 270 an arginine residue (R) is replaced byanother amino acid residue. Said amino acid residue is preferably anaromatic, acidic or basic amino acid residue. More preferably, saidamino acid residue is an aliphatic amino acid residue which isparticularly preferred to be a cysteine (C). The resulting polypeptideis shown in SEQ ID NO: 9.

At position 568 in exon 13 of the wild-type amino acid residue of P2X7Rdepicted in SEQ ID NO: 3 or 4 an isoleucine (I) residue is replaced byanother amino acid residue. More preferably, said isoleucine is replacedby an aromatic, basic or acidic amino acid residue. Even more preferred,said isoleucine is replaced by an aliphatic amino acid residue which isparticularly preferred to be an asparagine (N). The resultingpolypeptide is shown in SEQ ID NO: 10.

In exon 13 at position 578 in the wild-type amino acid sequence of P2X7Rdepicted in SEQ ID NO: 3 or 4 an argine residue (R) is replaced byanother amino acid residue. Said amino acid residue is preferably anaromatic, acidic or basic amino acid residue. More preferably, it is analiphatic amino acid residue and particularly preferred it is aglutamine (Q) residue. The resulting polypeptide is shown in SEQ ID NO:12.

It is envisaged that the above-mentioned mutations in the exons of theP2X7R gene occur due to point mutations caused by, e.g. chemical and/orphysical means or inaccuracy of the replication complex followed by afailure of the reparation machinery of a cell, can result in a change ofa single codon. Possible types of point mutations are transitions, i.e.change of a purine or pyrimidine base for another purine or pyrimidinebase, e.g. adenine to guanine or thymidine to cytosine or transversions,i.e. change of a purine or pyrimidine base for another pyrimide orpurine base, e.g., adenine to thymidine or guanine to cytosine.Additionally a point mutation can also be caused by insertion ordeletion of one or more nucleotides.

The mutations leading to the replacement of the amino acids as mentionedhereinabove and hereinbelow are indicated in Table 1 hereinbelow.

The third group of mutations in the P2X7R gene has been identified to bein exons 5 and 8 of the P2X7R gene depicted in SEQ ID NO: 1 and to besilent, i.e. they do not lead to amino acid changes. In particular, atposition 32548 in exon 5 of the wild-type genomic sequence P2X7R genedepicted in SEQ ID NO: 1 a cytidine residue is replaced by anothernucleotide. Said nucleotide is preferably a pyrimidine base andparticularly preferred a thymine. The exchange of the cytidine residueat position 32548 in exon 5 of the P2X7R gene by another nucleotidepreferably does not lead to the replacement of the amino acid cysteineby another amino acid residue.

In exon 8 of the wild-type P2X7R gene depicted in SEQ ID NO: 1 atposition 37633 a cytidine residue is replaced by another nucleotideresidue. Said nucleotide residue is preferably a pyrimidine base andparticularly preferred thymine. Due to this replacement the amino acidaspartate (D) encoded by the respective codon in which at position 37633a replacement has taken place is preferably not replaced by anotheramino acid residue.

The above-mentioned mutations in exons 5 and 8 at positions 32548 and37633, respectively, of the wild-type P2X7R gene depicted in SEQ ID NO:1are mutations at the third position of a triplet codon, i.e. at thewobble base, which lead to so-called silent mutations. Silent mutationsdo normally not lead to a change of the amino acid due to the degeneracyof the genetic code, i.e. 64 triplets encode at all 20 naturallyoccurring amino acids. However, said silent mutations lead to a changein the codon encoding its respective amino acids insofar that the newlygenerated codon may not fit so well into the codon usage of an organism.Namely, the newly generated codon is not translated by the ribosome withthe same efficiency as the “old” codon. This may lead to insufficientamounts of the corresponding polypeptide causing an distinct phenotype.

The fourth group of mutations in the P2X7R gene described hereinabove initem (d) is a deletion of 7 amino acids corresponding to positions 488to 494 of the wild-type P2X7R amino acid sequence as depicted in SEQ IDNO: 3 or 4. Thus, the present invention also relates to nucleic acidsequences encoding a P2X7R protein in which amino acids corresponding topositions 488 to 494 of the wild-type ATP-gated ion channel P2X7R asdepicted in SEQ ID NO: 3 or 4 are deleted. This means, according to thepresent invention, that a fragment encompassing amino acid positions 488to 494 of the corresponding wild-type amino acid sequence depicted inSEQ ID NO: 3 or 4 is deleted which results in a shortened polypeptide.An example for such a shortened polypeptide is depicted in SEQ ID NO:11. This type of mutation as described herein preferably encodes anon-functional ATP-gated ion channel P2X7R. In the present invention thedeletion of a fragment encompassing amino acids 488 to 494 of thewild-type amino acid sequence depicted in SEQ ID NO: 3 or 4 is theresult of a deletion in exon 13. The resulting protein depicted in SEQID NO: 11 lacks amino acids 488 to 494 of the corresponding wild typeamino acid sequence depicted in SEQ ID NO: 3 or 4 such that amino acidposition 494 of the deleted polypeptide depicted in SEQ ID NO: 11corresponds to amino acid position 502 of the wild-type amino acidsequence depicted in SEQ ID NO: 3 or 4. Preferably, the nucleic acidsequence of the invention encodes a P2X7R polypeptide in which exactlyamino acids corresponding to positions 488 to 494 of SEQ ID NO: 3 or 4are deleted. However, also mutants are comprised in which either more orless amino acids within the P2X7R amino acid sequence set forth in SEQID NO: 3 or 4 may be deleted due to, for example, atypical splicing ordeletion of nucleotides of the nucleic acid molecule encoding P2X7R orwrong posttranslational processes, as long as the P2X7R ATP-gated ionchannel is non-functional. For example, it is also possible that furtheramino acids preceding amino acid position 488 or amino acids succeedingamino acid position 494 may be deleted or that less amino acids aredeleted.

Preferably at least one, more preferably at least two, even morepreferably at least three and most preferably at least 5 amino acidresidues are further deleted upstream from the position corresponding toamino acid residue 488 and/or downstream of the position correspondingto amino acid residue 494 of SEQ ID NO: 3 or 4.

However, it is preferred that not more than 20, preferably not more than15, even more preferably not more than 10 and most preferably not morethan 7 amino acid residues are further deleted upstream of the positioncorresponding to amino acid residue 488 of SEQ ID NO: 3 or 4 ordownstream of the position corresponding to amino acid residue 494 ofSEQ ID NO: 3 or 4.

Another group of mutation (mentioned in item (e), supra) resides inintrons 1, 3, 4, 5, 6, 7, 9, 11 or 12 of the wild-type genomic sequenceof P2X7R depicted in SEQ ID NO: 1. Said mutations in said introns arepoint mutations as shown in Table B hereinabove and in Table 1,hereinbelow.

At the respective position indicated in the column “Position inwild-type” in Table B or indicated in the column “Polymorphism” in Table1 the position of the nucleotide residue in the respective intron whichis replaced by another nucleotide residue is shown. Accordingly, theterm “a nucleotide as indicated in column “Intron” of the Table Bcorresponding to the position as indicated in column “Replacednucleotide” of Table B corresponding to the position as indicated incolumn “Position in wild-type” of Table B is replaced by anothernucleotide means that a nucleotide residue in a P2X7R encoding sequencewould be located at position Y in SEQ ID NO: 1 when the P2X7R sequenceis compared and aligned with the sequence of SEQ ID NO: 1.

If the nucleotide at the respective position is a purine base such asadenine or guanine it is preferred that due to a transition it isreplaced by another purine base.

For example, an adenine is replaced by a guanine or a guanine isreplaced by an adenine. If the nucleotide at the respective position isa pyrimidine base it is preferred that due to a transition it isreplaced by another pyrimidine base. For example, thymine is replaced bya cytidine and a cytidine is replaced by a thymine.

It is also preferred that due to a transversion a purine base isreplaced by a pyrimidine base or vice versa. For example, an adenine isreplaced by a thymine and a guanine is replaced by a cytidine.Particularly preferred, said nucleotide in introns 1, 3, 4, 5, 6, 7, 9,11 or 12 of the P2X7R gene depicted in SEQ ID NO: 1 is replaced by thenucleotide depicted in column “Polymorphism” of Table 1, hereinbelow.

A last group of mutations that has been identified relates to mutationswhich reside in the 3′UTR of the wild-type P2X7R gene depicted in SEQ IDNO: 1. The mutations were found at positions 54925, 55169, 55170, 55171or 55917 respectively, of the wild-type P2X7R gene depicted in SEQ IDNO: 1.

At position 54925 a guanine residue was found to be replaced by anothernucleotide. Preferably, said guanine residue is replaced by a pyrimidinebase, more preferably by a purine base and particularly preferred by anadenine.

At position 55169 a cytidine residue is replaced by another nucleotide,preferably by a pyrimidine base. More preferably, it is replaced by apurine base and particularly preferred, it is replaced by an adenine.

At positions 55170 and 55171 an adenine residue is replaced by anothernucleotide residue, preferably by a purine base. More preferably, saidadenine residue is replaced by a pyrimidine base and particularlypreferred said adenine residue is replaced by a cytidine residue. It wasalso found that at position 55917 a cytidine residue is replaced byanother nucleotide. Preferably, said nucleotide residue is a purinebase, more preferably a pyrimidine base and particularly preferable athymine.

As is evident from the above, not all identified mutations are locatedin exons or lead to a change in the amino acid sequence. Some of themutations are located in the 5′UTR, the 3′UTR or in introns.

It is known that polymorphisms in promoter and enhancer regions canaffect gene function by modulating transcription, particularly if theyare situated at recognition sites for DNA binding proteins (Fishman etal., J. Clin. Invest. 102 (1998), 1369-1376). The term “polymorphism”which is used in the present invention means single nucleotidesubstitution, nucleotide insertion and nucleotide deletion which in thecase of insertion and deletion includes insertion or deletion of one ormore nucleotides at a position of a gene and corresponding alterationsin expressed proteins. Polymorphisms in the 5′ untranslated region(5′UTR) of genes can affect the efficiency with which proteins aretranslated. A representative example of this is in the c-myc gene wherea C-G SNP that creates an internal ribosome entry site is associatedwith increased efficiency of c-myc translation and myeloma (Chappell etal., Oncogene 19 (2000), 4437-4440). Polymorphisms in the 3′UTR canaffect gene function by altering the secondary structure of RNA andefficiency of translation or by affecting motifs in the RNA that bindproteins which regulate RNA degradation. Polymorphisms within intronscan affect gene function by affecting RNA splicing resulting in aberrantpolypeptides. Another way in which intronic polymorphisms can affectgene function is when they affect regulatory motifs within introns.Examples are the Sp1 binding site polymorphism within intron 1 of theCOLIA1 gene (Mann et al., J. Clin. Invest 107 (2001), 899-907) and arepeat polymorphisms within the IL-1Ra gene (Keen et al., Bone 23(1998), 367-371). Further examples between intronic SNPs and genefunction are described in Caceres and Komblihtt, Trends Genet. 4 (2002),186-93. Example 4 on page 52, line 30 to page 53, line 51 of the textdescribes potential alternative splicing events and aberrant proteinproduction associated with three SNPs disclosed in the application.

The nucleic acid sequences described hereinabove may comprise at least56580 nucleotides, preferably at least 10000 nucleotides, at least 5000nucleotides, at least 1000 nucleotides, at least 500 nucleotides, atleast 100 nucleotides. More preferably, said nucleic acid sequencescomprise at least 50 nucleotides and particularly preferred theycomprise at least 20 or 21 nucleotides comprising the mutations ordeletions as described hereinabove. Most preferably such a nucleic acidsequence has a sequence as depicted in any one of SEQ ID NOs: 13 to 51.

The nucleic acid sequences described hereinabove which comprisemutations in exons leading to a replacement of the corresponding aminoacid sequence of the P2X7R wild-type polypeptide depicted in SEQ ID NO:3 or 4 encode polypeptides shown in SEQ ID NOs: 5 to 10 and 12

Additionally, the nucleic acid sequences described hereinabove whichcomprise a deletion leading to a truncated polypeptide in comparison tothe full-length polypeptide of the wild-type P2X7R polypeptide shown inSEQ ID NO: 3 or 4 is shown in SEQ ID NO: 11.

The present invention also relates to nucleic acid molecules whichhybridize to one of the above described nucleic acid molecules and whichshows a mutation as described hereinabove.

The term “hybridizes” as used in accordance with the present inventionmay relate to hybridizations under stringent or non-stringentconditions. If not further specified, the conditions are preferablynon-stringent. Said hybridization conditions may be establishedaccording to conventional protocols described, for example, in Sambrook,Russell “Molecular Cloning, A Laboratory Manual”, Cold Spring HarborLaboratory, N.Y. (2001); Ausubel, “Current Protocols in MolecularBiology”, Green Publishing Associates and Wiley Interscience, N.Y.(1989), or Higgins and Hames (Eds.) “Nucleic acid hybridization, apractical approach” IRL Press Oxford, Washington D.C., (1985). Thesetting of conditions is well within the skill of the artisan and can bedetermined according to protocols described in the art. Thus, thedetection of only specifically hybridizing sequences will usuallyrequire stringent hybridization and washing conditions such as 0.1×SSC,0.1% SDS at 65° C. Non-stringent hybridization conditions for thedetection of homologous or not exactly complementary sequences may beset at 6×SSC, 1% SDS at 65° C. As is well known, the length of the probeand the composition of the nucleic acid to be determined constitutefurther parameters of the hybridization conditions. Note that variationsin the above conditions may be accomplished through the inclusion and/orsubstitution of alternate blocking reagents used to suppress backgroundin hybridization experiments. Typical blocking reagents includeDenhardt's reagent, BLOTTO, heparin, denatured salmon sperm DNA, andcommercially available proprietary formulations. The inclusion ofspecific blocking reagents may require modification of the hybridizationconditions described above, due to problems with compatibility.Hybridizing nucleic acid molecules also comprise fragments of the abovedescribed molecules. Such fragments may represent nucleic acid sequenceswhich code for a non-functional ATP-gated ion channel P2X7R or anon-functional fragment thereof, and which have a length of at least 12nucleotides, preferably at least 15, more preferably at least 18, morepreferably of at least 21 nucleotides, more preferably at least 30nucleotides, even more preferably at least 40 nucleotides and mostpreferably at least 60 nucleotides. Furthermore, nucleic acid moleculeswhich hybridize with any of the aforementioned nucleic acid moleculesalso include complementary fragments, derivatives and allelic variantsof these molecules. Additionally, a hybridization complex refers to acomplex between two nucleic acid sequences by virtue of the formation ofhydrogen bonds between complementary G and C bases and betweencomplementary A and T bases; these hydrogen bonds may be furtherstabilized by base stacking interactions. The two complementary nucleicacid sequences hydrogen bond in an antiparallel configuration. Ahybridization complex may be formed in solution (e.g., Cot or Rotanalysis) or between one nucleic acid sequence present in solution andanother nucleic acid sequence immobilized on a solid support (e.g.,membranes, filters, chips, pins or glass slides to which, e.g., cellshave been fixed). The terms complementary or complementarity refer tothe natural binding of polynucleotides under permissive salt andtemperature conditions by base-pairing. For example, the sequence“A-G-T” binds to the complementary sequence “T-C-A”. Complementaritybetween two single-stranded molecules may be “partial”, in which onlysome of the nucleic acids bind, or it may be complete when totalcomplementarity exists between single-stranded molecules. The degree ofcomplementarity between nucleic acid strands has significant effects onthe efficiency and strength of hybridization between nucleic acidstrands. This is of particular importance in amplification reactions,which depend upon binding between nucleic acids strands.

The term “hybridizing sequences” preferably refers to sequences whichdisplay a sequence identity of at least 40%, preferably at least 50%,more preferably at least 60%, even more preferably at least 70%,particularly preferred at least 80%, more particularly preferred atleast 90%, even more particularly preferred at least 95% and mostpreferably at least 97% identity with a nucleic acid sequence asdescribed above encoding a P2X7R protein having a described mutation.Moreover, the term “hybridizing sequences” preferably refers tosequences encoding a P2X7R protein having a sequence identity of atleast 40%, preferably at least 50%, more preferably at least 60%, evenmore preferably at least 70%, particularly preferred at least 80%, moreparticularly preferred at least 90%, even more particularly preferred atleast 95% and most preferably at least 97% identity with an amino acidsequence of a P2X7R mutant as described herein above.

In accordance with the present invention, the term “identical” or“percent identity” in the context of two or more nucleic acid or aminoacid sequences, refers to two or more sequences or subsequences that arethe same, or that have a specified percentage of amino acid residues ornucleotides that are the same (e.g., 60% or 65% identity, preferably,70-95% identity, more preferably at least 95% identity), when comparedand aligned for maximum correspondence over a window of comparison, orover a designated region as measured using a sequence comparisonalgorithm as known in the art, or by manual alignment and visualinspection. Sequences having, for example, 60% to 95% or greatersequence identity are considered to be substantially identical. Such adefinition also applies to the complement of a test sequence. Preferablythe described identity exists over a region that is at least about 15 to25 amino acids or nucleotides in length, more preferably, over a regionthat is about 50 to 100 amino acids or nucleotides in length. Thosehaving skill in the art will know how to determine percent identitybetween/among sequences using, for example, algorithms such as thosebased on CLUSTALW computer program (Thompson Nucl. Acids Res. 2 (1994),46734680) or FASTDB (Brutlag Comp. App. Biosci. 6 (1990), 237-245), asknown in the art.

Although the FASTDB algorithm typically does not consider internalnon-matching deletions or additions in sequences, i.e., gaps, in itscalculation, this can be corrected manually to avoid an overestimationof the % identity. CLUSTALW, however, does take sequence gaps intoaccount in its identity calculations. Also available to those havingskill in this art are the BLAST and BLAST 2.0 algorithms (Altschul Nucl.Acids Res. 25 (1977), 3389-3402). The BLASTN program for nucleic acidsequences uses as defaults a word length (W) of 11, an expectation (E)of 10, M=5, N=4, and a comparison of both strands. For amino acidsequences, the BLASTP program uses as defaults a wordlength (W) of 3,and an expectation (E) of 10. The BLOSUM62 scoring matrix (HenikoffProc. Natl. Acad. Sci., USA, 89, (1989), 10915) uses alignments (B) of50, expectation (E) of 10, M=5, N=4, and a comparison of both strands.

Moreover, the present invention also relates to nucleic acid moleculesthe sequence of which is degenerate in comparison with the sequence ofan above-described hybridizing molecule. When used in accordance withthe present invention the term “being degenerate as a result of thegenetic code” means that due to the redundancy of the genetic codedifferent nucleotide sequences code for the same amino acid.

The present invention also related to nucleic acid molecules whichcomprise one or more of the above-described mutations or deletions.

The nucleic acid molecules according to the invention may be derivedfrom any organism encoding corresponding P2X7R ATP-gated ion channels.For example, P2X7R ATP-gated ion channels have been reported in variousorganisms, for example, rat (see, Suprenant (1996), loc. cit.), mouse(Genbank Accession No. AJ 489297), xenopus (Genbank Accession No. AJ345114), chicken (Genbank Accession No. BM 491404) or Bos Taurus(Genbank Accession No. AF 083073). In a preferred embodiment the nucleicacid molecule of the invention is derived from a vertebrate, preferablyfrom a mammal, even more preferably the nucleic acid molecule is derivedfrom rabbit or guinea pig, and most preferably the nucleic acid isderived from mouse, rat or human.

The nucleic acid molecule according to the invention may be any type ofnucleic acid, e.g. DNA, RNA or PNA (peptide nucleic acid).

For the purposes of the present invention, a peptide nucleic acid (PNA)is a polyamide type of DNA analog and the monomeric units for adenine,guanine, thymine and cytosine are available commercially (PerceptiveBiosystems). Certain components of DNA, such as phosphorus, phosphorusoxides, or deoxyribose derivatives, are not present in PNAs. Asdisclosed by Nielsen et al., Science 254:1497 (1991); and Egholm et al.,Nature 365:666 (1993), PNAs bind specifically and tightly tocomplementary DNA strands and are not degraded by nucleases. In fact,PNA binds more strongly to DNA than DNA itself does. This is probablybecause there is no electrostatic repulsion between the two strands, andalso the polyamide backbone is more flexible. Because of this, PNA/DNAduplexes bind under a wider range of stringency conditions than DNA/DNAduplexes, making it easier to perform multiplex hybridization. Smallerprobes can be used than with DNA due to the strong binding. In addition,it is more likely that single base mismatches can be determined withPNA/DNA hybridization because a single mismatch in a PNA/DNA 15-merlowers the melting point (T.sub.m) by 8°-20° C., vs. 4°-16° C. for theDNA/DNA 15-mer duplex. Also, the absence of charge groups in PNA meansthat hybridization can be done at low ionic strengths and reducepossible interference by salt during the analysis.

The DNA may, for example, be cDNA. In a preferred embodiment it is agenomic DNA. The RNA may be, e.g., mRNA. The nucleic acid molecule maybe natural, synthetic or semisynthetic or it may be a derivative, suchas peptide nucleic acid (Nielsen, Science 254 (1991), 1497-1500) orphosphorothioates. Furthermore, the nucleic acid molecule may be arecombinantly produced chimeric nucleic acid molecule comprising any ofthe aforementioned nucleic acid molecules either alone or incombination.

Preferably, the nucleic acid molecule of the present invention is partof a vector. Therefore, the present invention relates in anotherembodiment to a vector comprising the nucleic acid molecule of thisinvention. Such a vector may be, e.g., a plasmid, cosmid, virus,bacteriophage or another vector used e.g. conventionally in geneticengineering, and may comprise further genes such as marker genes whichallow for the selection of said vector in a suitable host cell and undersuitable conditions.

The nucleic acid molecules of the present invention may be inserted intoseveral commercially available vectors. Nonlimiting examples includeplasmid vectors compatible with mammalian cells, such as pUC,pBluescript (Stratagene), pET (Novagen), pREP (Invitrogen), pCRTopo(Invitrogen), pcDNA3 (Invitrogen), pCEP4 (Invitrogen), pMC1 neo(Stratagene), pXT1 (Stratagene), pSG5 (Stratagene), EBO-pSV2neo, pBPV-1,pdBPVMMTneo, pRSVgpt, pRSVneo, pSV2-dhfr, pUCTag, pIZD35, pLXIN and pSIR(Clontech) and pIRES-EGFP (Clontech). Baculovirus vectors such aspBlueBac, BacPacz Baculovirus Expression System (CLONTECH), and MaxBac™Baculovirus Expression System, insect cells and protocols (Invitrogen)are available commercially and may also be used to produce high yieldsof biologically active protein. (see also, Miller (1993), Curr. Op.Genet. Dev., 3, 9; O'Reilly, Baculovirus Expression Vectors: ALaboratory Manual, p. 127). In addition, prokaryotic vectors such aspcDNA2; and yeast vectors such as pYes2 are nonlimiting examples ofother vectors suitable for use with the present invention. For vectormodification techniques, see Sambrook and Russel (2001), loc. cit.Vectors can contain one or more replication and inheritance systems forcloning or expression, one or more markers for selection in the host,e.g., antibiotic resistance, and one or more expression cassettes.

The coding sequences inserted in the vector can be synthesized bystandard methods, isolated from natural sources, or prepared as hybrids.Ligation of the coding sequences to transcriptional regulatory elements(e.g., promoters, enhancers, and/or insulators) and/or to other aminoacid encoding sequences can be carried out using established methods.

Furthermore, the vectors may, in addition to the nucleic acid sequencesof the invention, comprise expression control elements, allowing properexpression of the coding regions in suitable hosts. Such controlelements are known to the artisan and may include a promoter,translation initiation codon, translation and insertion site or internalribosomal entry sites (IRES) (Owens, Proc. Natl. Acad. Sci. USA 98(2001), 1471-1476) for introducing an insert into the vector.Preferably, the nucleic acid molecule of the invention is operativelylinked to said expression control sequences allowing expression ineukaryotic or prokaryotic cells. Particularly preferred are in thiscontext control sequences which allow for correct expression in neuronalcells and/or cells derived from nervous tissue.

Control elements ensuring expression in eukaryotic and prokaryotic cellsare well known to those skilled in the art. As mentioned above, theyusually comprise regulatory sequences ensuring initiation oftranscription and optionally poly-A signals ensuring termination oftranscription and stabilization of the transcript. Additional regulatoryelements may include transcriptional as well as translational enhancers,and/or naturally-associated or heterologous promoter regions. Possibleregulatory elements permitting expression in for example mammalian hostcells comprise the CMV-HSV thymidine kinase promoter, SV40, RSV-promoter(Rous sarcome virus), human elongation factor 1α-promoter, CMV enhancer,CaM-kinase promoter or SV40-enhancer.

For the expression for example in nervous tissue and/or cells derivedtherefrom, several regulatory sequences are well known in the art, likethe minimal promoter sequence of human neurofilament L (Charron, J.Biol. Chem. 270 (1995), 25739-25745). For the expression in prokaryoticcells, a multitude of promoters including, for example, thetac-lac-promoter, the lacUV5 or the trp promoter, has been described.Beside elements which are responsible for the initiation oftranscription such regulatory elements may also comprise transcriptiontermination signals, such as SV40-poly-A site or the tk-poly-A site,downstream of the polynucleotide. In this context, suitable expressionvectors are known in the art such as Okayama-Berg cDNA expression vectorpcDV1 (Pharmacia), pRc/CMV, pcDNA1, pcDNA3 (In-Vitrogene, as used, interalia in the appended examples), pSPORT1 (GIBCO BRL) or pGEMHE (Promega),or prokaryotic expression vectors, such as lambda gt11.

An expression vector according to this invention is at least capable ofdirecting the replication, and preferably the expression, of the nucleicacids and protein of this invention. Suitable origins of replicationinclude, for example, the Col E1, the SV40 viral and the M 13 origins ofreplication. Suitable promoters include, for example, thecytomegalovirus (CMV) promoter, the lacZ promoter, the gai10 promoterand the Autographa californica multiple nuclear polyhedrosis virus(AcMNPV) polyhedral promoter. Suitable termination sequences include,for example, the bovine growth hormone, SV40, lacZ and AcMNPV polyhedralpolyadenylation signals. Examples of selectable markers includeneomycin, ampicillin, and hygromycin resistance and the like.Specifically-designed vectors allow the shuttling of DNA betweendifferent host cells, such as bacteria-yeast, or bacteria-animal cells,or bacteria-fungal cells, or bacteria invertebrate cells.

Beside the nucleic acid molecules of the present invention, the vectormay further comprise nucleic acid sequences encoding for secretionsignals. Such sequences are well known to the person skilled in the art.Furthermore, depending on the expression system used leader sequencescapable of directing the expressed polypeptide to a cellular compartmentmay be added to the coding sequence of the nucleic acid molecules of theinvention and are well known in the art. The leader sequence(s) is (are)assembled in appropriate phase with translation, initiation andtermination sequences, and preferably, a leader sequence capable ofdirecting secretion of translated protein, or a part thereof, into,inter alia, the extracellular membrane. Optionally, the heterologoussequence can encode a fusion protein including an C- or N-terminalidentification peptide imparting desired characteristics, e.g.,stabilization or simplified purification of expressed recombinantproduct. Once the vector has been incorporated into the appropriatehost, the host is maintained under conditions suitable for high levelexpression of the nucleotide sequences, and, as desired, the collectionand purification of the proteins, antigenic fragments or fusion proteinsof the invention may follow. Of course, the vector can also compriseregulatory regions from pathogenic organisms.

Furthermore, said vector may also be, besides an expression vector, agene transfer and/or gene targeting vector. Gene therapy, which is basedon introducing therapeutic genes (for example for vaccination) intocells by ex-vivo or in-vivo techniques is one of the most importantapplications of gene transfer. Suitable vectors, vector systems andmethods for in-vitro or in-vivo gene therapy are described in theliterature and are known to the person skilled in the art; see, e.g.,Giordano, Nature Medicine 2 (1996), 534-539; Schaper, Circ. Res. 79(1996), 911-919; Anderson, Science 256 (1992), 808-813, Isner, Lancet348 (1996), 370-374; Muhlhauser, Circ. Res. 77 (1995), 1077-1086; Wang,Nature Medicine 2 (1996), 714-716; WO 94/29469; WO 97/00957; Schaper,Current Opinion in Biotechnology 7 (1996), 635-640 or Verma, Nature 389(1997), 239-242 and references cited therein. The nucleic acid moleculesof the invention and vectors as described herein above may be designedfor direct introduction or for introduction via liposomes, or viralvectors (e.g. adenoviral, retroviral) into the cell. Additionally,baculoviral systems or systems based on vaccinia virus or Semliki ForestVirus can be used as eukaryotic expression system for the nucleic acidmolecules of the invention. In addition to recombinant production,fragments of the protein, the fusion protein or antigenic fragments ofthe invention may be produced by direct peptide synthesis usingsolid-phase techniques (cf Stewart et al. (1969) Solid Phase PeptideSynthesis; Freeman Co, San Francisco; Merrifield, J. Am. Chem. Soc. 85(1963), 2149-2154). In vitro protein synthesis may be performed usingmanual techniques or by automation. Automated synthesis may be achieved,for example, using Applied Biosystems 431A Peptide Synthesizer (PerkinElmer, Foster City Calif.) in accordance with the instructions providedby the manufacturer. Various fragments may be chemically synthesizedseparately and combined using chemical methods to produce the fulllength molecule.

The present invention in addition relates to a host transformed with avector of the present invention or to a host comprising the nucleic acidmolecule of the invention. Said host may be produced by introducing saidvector or nucleotide sequence into a host cell which upon its presencein the cell mediates the expression of a protein encoded by thenucleotide sequence of the invention or comprising a nucleotide sequenceor a vector according to the invention wherein the nucleotide sequenceand/or the encoded polypeptide is foreign to the host cell.

By “foreign” it is meant that the nucleotide sequence and/or the encodedpolypeptide is either heterologous with respect to the host, this meansderived from a cell or organism with a different genomic background, oris homologous with respect to the host but located in a differentgenomic environment than the naturally occurring counterpart of saidnucleotide sequence. This means that, if the nucleotide sequence ishomologous with respect to the host, it is not located in its naturallocation in the genome of said host, in particular it is surrounded bydifferent genes. In this case the nucleotide sequence may be eitherunder the control of its own promoter or under the control of aheterologous promoter. The location of the introduced nucleic acidmolecule or the vector can be determined by the skilled person by usingmethods well-known to the person skilled in the art, e.g., SouthernBlotting. The vector or nucleotide sequence according to the inventionwhich is present in the host may either be integrated into the genome ofthe host or it may be maintained in some form extrachromosomally. Inthis respect, it is also to be understood that the nucleotide sequenceof the invention can be used to restore or create a mutant gene viahomologous recombination.

Said host may be any prokaryotic or eukaryotic cell. Suitableprokaryotic/bacterial cells are those generally used for cloning like E.coli, Salmonella typhimurium, Serratia marcescens or Bacillus subtilis.Said eukaryotic host may be a mammalian cell, an amphibian cell, a fishcell, an insect cell, a fungal cell, a plant cell or a bacterial cell(e.g., E coli strains HB101, DH5a, XL1 Blue, Y1090 and JM101).Eukaryotic recombinant host cells are preferred. Examples of eukaryotichost cells include, but are not limited to, yeast, e.g., Saccharomycescerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis or Pichiapastoris cells, cell lines of human, bovine, porcine, monkey, and rodentorigin, as well as insect cells, including but not limited to,Spodoptera frugiperda insect cells and Drosophila-derived insect cellsas well as zebra fish cells. Mammalian species-derived cell linessuitable for use and commercially available include, but are not limitedto, L cells, CV-1 cells, COS-1 cells (ATCC CRL 1650), COS-7 cells (ATCCCRL 1651), HeLa cells (ATCC CCL 2), C1271 (ATCC CRL 1616), BS-C-1 (ATCCCCL 26) and MRC-5 (ATCC CCL 171).

In a particularly preferred embodiment said mammalian cell is a neuronalcell and/or a cultured cell like, inter alia, a HEK 293 (human embryonickidney) cell, a CHO, HeLa, NIH3T3, BHK, PC12 cell or a neuronal stemcell preferably derived from a mammal and more preferably from a human.

In another more preferred embodiment said amphibian cell is an oocyte.In an even more preferred embodiment said oocyte is a frog oocyte,particularly preferred a Xenopus laevis oocyte.

In a more preferred embodiment, the host according to the invention is anon-human transgenic organism. Said non-human organism may be a mammal,amphibian, a fish, an insect, a fungus or a plant. Particularlypreferred non-human transgenic animals are Drosophila species,Caenorhabditis elegans, Xenopus species, zebra fish, Spodopterafrugiperda, Autographa californica, mice and rats. Transgenic plantscomprise, but are not limited to, wheat, tobacco, parsley andArabidopsis. Transgenic fungi are also well known in the art andcomprise, inter alia, yeasts, like S. pombe or S. cerevisae, orAspergillus, Neurospora or Ustilago species or Pichia species.

In another embodiment, the present invention relates to a method forproducing the polypeptide encoded by a nucleic acid molecule of theinvention comprising culturing/raising the host of the invention andisolating the produced polypeptide. A large number of suitable methodsexist in the art to produce polypeptides in appropriate hosts. If thehost is a unicellular organism or a mammalian or insect cell, the personskilled in the art can revert to a variety of culture conditions thatcan be further optimized without an undue burden of work. Conveniently,the produced protein is harvested from the culture medium or fromisolated (biological) membranes by established techniques. Furthermore,the produced polypeptide may be directly isolated from the host cell.Said host cell may be part of or derived from a part of a host organism,for example said host cell may be part of the CNS of an animal or theharvestable part of a plant. Additionally, the produced polypeptide maybe isolated from fluids derived from said host, like blood, milk orcerebrospinal fluid.

Additionally the present invention relates to polypeptides depicted inSEQ ID NOs: 5 to 12 which are encoded by the nucleic acid molecules ofthe invention or produced by the method of the invention. Thepolypeptide of the invention may accordingly be produced bymicrobiological methods or by transgenic mammals. It is also envisagedthat the polypeptide of the invention is recovered from transgenicplants. Alternatively, the polypeptide of the invention may be producedsynthetically or semi-synthetically.

For example, chemical synthesis, such as the solid phase proceduredescribed by Houghton Proc. Natl. Acad. Sci. USA (82) (1985), 5131-5135,can be used. Another method is in vitro translation of mRNA. A preferredmethod involves the recombinant production of protein in host cells asdescribed above. For example, nucleotide acid sequences comprising allor a portion of any one of the nucleotide sequences according to theinvention can be synthesized by PCR, inserted into an expression vector,and a host cell transformed with the expression vector. Thereafter, thehost cell is cultured to produce the desired polypeptide, which isisolated and purified. Protein isolation and purification can beachieved by any one of several known techniques; for example and withoutlimitation, ion exchange chromatography, gel filtration chromatographyand affinity chromatography, high pressure liquid chromatography (HPLC),reversed phase HPLC, preparative disc gel electrophoresis. In addition,cell-free translation systems can be used to produce the polypeptides ofthe present invention. Suitable cell-free expression systems for use inaccordance with the present invention include rabbit reticulocytelysate, wheat germ extract, canine pancreatic microsomal membranes, E.coli S30 extract, and coupled transcription/translation systems such asthe TNT-system (Promega). These systems allow the expression ofrecombinant polypeptides or peptides upon the addition of cloningvectors, DNA fragments, or RNA sequences containing coding regions andappropriate promoter elements. As mentioned supra, proteinisolation/purification techniques may require modification of theproteins of the present invention using conventional methods. Forexample, a histidine tag can be added to the protein to allowpurification on a nickel column. Other modifications may cause higher orlower activity, permit higher levels of protein production, or simplifypurification of the protein.

In a further embodiment, the present invention relates to an antibodyspecifically directed to a polypeptide of the invention, wherein saidantibody specifically reacts with an epitope generated and/or formed bythe mutation in the ATP-gated ion channel P2X7R selected from the groupconsisting of:

-   (i) an epitope specifically presented by a polypeptide which has an    amino acid sequence of an ATP-gated ion channel P2X7R, wherein the R    (Arg), G (Gly), E (Glu), L (Leu), R (Arg), I (Ile) or R (Arg)    residue corresponding to position 117, 150, 186, 191, 270, 568 or    578 of the wild-type ATP-gated ion channel P2X7R as depicted in SEQ    ID NO: 3 or 4 is replaced by another amino acid residue; and-   (ii) an epitope specifically presented by a polypeptide which has an    amino acid sequence of an ATP-gated ion channel P2X7R, wherein amino    acids corresponding to positions 488 to 494 of the wild-type    ATP-gated ion channel P2X7R as depicted in SEQ ID NO: 3 or 4 are    deleted.

With respect to preferred embodiments of (i) and (ii) the same appliesas described above in connection with the nucleic acid molecules. Theterm “specifically” in this context means that the antibody reacts withthe mutant P2X7R protein but not with a wild-type P2X7R protein.Preferably this term also means that such an antibody does not bind toother mutant forms of the P2X7R protein, in particular those describedherein. Whether the antibody specifically reacts as defined herein abovecan easily be tested, inter alia, by comparing the reaction of saidantibody with a wild-type ATP-gated ion channel P2X7R (or a subunit or afragment thereof with the reaction of said antibody with a mutant P2X7Rpolypeptide of the invention.

The antibody of the present invention can be, for example, polyclonal ormonoclonal. The term “antibody” also comprises derivatives or fragmentsthereof which still retain the binding specificity. Techniques for theproduction of antibodies are well known in the art and described, e.g.in Harlow and Lane “Antibodies, A Laboratory Manual”, CSH Press, ColdSpring Harbor, 1988. These antibodies can be used, for example, for theimmunoprecipitation and immunolocalization of the polypeptides of theinvention as well as for the monitoring of the presence of suchpolypeptides, for example, in recombinant organisms or in diagnosis.They can also be used for the identification of compounds interactingwith the proteins according to the invention (as mentioned hereinbelow). For example, surface plasmon resonance as employed in theBIAcore system can be used to increase the efficiency of phageantibodies which bind to an epitope of the polypeptide of the invention(Schier, Human Antibodies Hybridomas 7 (1996), 97-105; Malmborg, J.Immunol. Methods 183 (1995), 7-13).

The present invention furthermore includes chimeric, single chain andhumanized antibodies, as well as antibody fragments, like, inter alia,Fab fragments. Antibody fragments or derivatives further compriseF(ab′)2, Fv or scFv fragments; see, for example, Harlow and Lane, loc.cit. Various procedures are known in the art and may be used for theproduction of such antibodies and/or fragments. Thus, the (antibody)derivatives can be produced by peptidomimetics. Further, techniquesdescribed for the production of single chain antibodies (see, interalia, U.S. Pat. No. 4,946,778) can be adapted to produce single chainantibodies to polypeptide(s) of this invention. Also, transgenic animalsmay be used to express humanized antibodies to polypeptides of thisinvention. Most preferably, the antibody of this invention is amonoclonal antibody. For the preparation of monoclonal antibodies, anytechnique which provides antibodies produced by continuous cell linecultures can be used. Examples for such techniques include the hybridomatechnique (Köhler and Milstein Nature 256 (1975), 495-497), the triomatechnique, the human B-cell hybridoma technique (Kozbor, ImmunologyToday 4 (1983), 72) and the EBV-hybridoma technique to produce humanmonoclonal antibodies (Cole et al., Monoclonal Antibodies and CancerTherapy, Alan R. Liss, Inc. (1985), 77-96). Techniques describing theproduction of single chain antibodies (e.g., U.S. Pat. No. 4,946,778)can be adapted to produce single chain antibodies to immunogenicpolypeptides as described above. Furthermore, transgenic mice may beused to express humanized antibodies directed against said immunogenicpolypeptides. It is in particular preferred that the antibodies/antibodyconstructs as well as antibody fragments or derivatives to be employedin accordance with this invention or capable to be expressed in a cell.This may, inter alia, be achieved by direct injection of thecorresponding proteineous molecules or by injection of nucleic acidmolecules encoding the same. Furthermore, gene therapy approaches areenvisaged. Accordingly, in context of the present invention, the term“antibody molecule” relates to full immunoglobulin molecules as well asto parts of such immunoglobulin molecules. Furthermore, the termrelates, as discussed above, to modified and/or altered antibodymolecules, like chimeric and humanized antibodies. The term also relatesto monoclonal or polyclonal antibodies as well as to recombinantly orsynthetically generated/synthesized antibodies. The term also relates tointact antibodies as well as to antibody fragments thereof, like,separated light and heavy chains, Fab, Fab/c, Fv, Fab′, F(ab′)2. Theterm “antibody molecule” also comprises bifunctional antibodies andantibody constructs, like single chain Fvs (scFv) or antibody-fusionproteins. It is also envisaged in context of this invention that theterm “antibody” comprises antibody constructs which may be expressed incells, e.g. antibody constructs which may be transfected and/ortransduced via, inter alia, viruses or vectors. It is in particularenvisaged that such antibody constructs specifically recognize thepolypeptides of the present invention. It is, furthermore, envisagedthat said antibody construct is employed in gene therapy approaches.

The present invention relates also to an aptamer specifically binding toa polypeptide according to the invention wherein said aptamer reactswith an epitope of a polypeptide of the present invention. The presentinvention furthermore relates to an aptamer specifically directed to acorresponding nucleic acid molecule according to the invention.

In accordance with the present invention, the term “aptamer” meansnucleic acid molecules that can bind to target molecules. Aptamerscommonly comprise RNA, single stranded DNA, modified RNA or modified DNAmolecules. The preparation of aptamers is well known in the art and mayinvolve, inter alia, the use of combinatorial RNA libraries to identifybinding sides (Gold, Ann. Rev. Biochem. 64 (1995), 763-797).

Furthermore, the present invention relates to a primer or pair ofprimers capable of specifically amplifying the nucleic acid molecules ofthe present invention. The term “primer” when used in the presentinvention means a single-stranded nucleic acid molecule capable ofannealing the nucleic acid molecule of the present application andthereby being capable of serving as a starting point for amplification.Said term also comprises oligoribo- or desoxyribonucleotides which arecomplementary to a region of one of the strands of a nucleic acidmolecule of the present invention. According to the present inventionthe term “pair of primers” means a pair of primers that are with respectto a complementary region of a nucleic acid molecule directed in theopposite direction towards each other to enable, for example,amplification by polymerase chain reaction (PCR).

The term “amplifying” refers to repeated copying of a specified sequenceof nucleotides resulting in an increase in the amount of said specifiedsequence of nucleotides. and allows the generation of a multitude ofidentical or essentially identical (i.e. at least 95% more preferred atleast 98%, even more preferred at least 99% and most preferred at least99.5% such as 99.9% identical) nucleic acid molecules or parts thereof.Such methods are well established in the art; see Sambrook et al.“Molecular Cloning, A Laboratory Manual”, 2^(nd) edition 1989, CSHPress, Cold Spring Harbor. They include polymerase chain reaction (PCR)and modifications thereof, ligase chain reaction (LCR) to name somepreferred amplification methods.

When used in the context of primers the term “specifically” means thatonly the nucleic acid molecules as described herein above are amplifiedand nucleic acid molecules encoding the wild-type P2X7R ATP-gatedreceptor as depicted in SEQ ID NO: 1 are not amplified. Thus, a primeraccording to the invention is preferably a primer which binds to aregion of a nucleic acid molecule of the invention which is unique forthis molecule and which is not present in the wild-type P2X7R encodingsequence, i.e. the primer binds in a region in which one of the abovedescribed mutations occur. In connection with a pair of primersaccording to the invention it is possible that one of the primers of thepair is specific in the above described meaning or both of the primersof the pair are specific. In both cases, the use of such a pair ofprimers would allow to specifically amplify a mutant of the invention asdescribed herein-above but not the wild-type P2X7R encoding sequence.

The 3′-OH end of a primer is used by a polymerase to be extended bysuccessive incorporation of nucleotides. The primer or pair of primersof the present invention can be used, for example, in primer extensionexperiments on template RNA according to methods known by the personskilled in the art. Preferably, the primer or pair of primers of thepresent invention are used for amplification reactions on template RNAor template DNA, preferably cDNA or genomic DNA. The terms “templateDNA” or “template RNA” refers to DNA or RNA molecules or fragmentsthereof of any source or nucleotide composition, that comprise a targetnucleotide sequence as defined above. The primer or pair of primers canalso be used for hybridization experiments as known in the art.Preferably, the primer or pair of primers are used in polymerase chainreactions to amplify sequences corresponding to a sequence of thenucleic acid molecule of the present invention. It is known that thelength of a primer results from different parameters (Gillam, Gene 8(1979), 81-97; Innis, PCR Protocols: A guide to methods andapplications, Academic Press, San Diego, USA (1990)). Preferably, theprimer should only hybridize or bind to a specific region of a targetnucleotide sequence. The length of a primer that statisticallyhybridizes only to one region of a target nucleotide sequence can becalculated by the following formula: (¼)^(x) (whereby x is the length ofthe primer). For example a hepta- or octanucleotide would be sufficientto bind statistically only once on a sequence of 37 kb. However, it isknown that a primer exactly matching to a complementary template strandmust be at least 9 base pairs in length, otherwise no stable-doublestrand can be generated (Goulian, Biochemistry 12 (1973), 2893-2901). Itis also envisaged that computer-based algorithms can be used to designprimers capable of amplifying the nucleic acid molecules of theinvention. Preferably, the primers of the invention are at least 10nucleotides in length, more preferred at least 12 nucleotides in length,even more preferred at least 15 nucleotides in length, particularlypreferred at least 18 nucleotides in length, even more particularlypreferred at least 20 nucleotides in length and most preferably at least25 nucleotides in length. The invention, however, can also be carriedout with primers which are shorter or longer.

It is also envisaged that the primer or pair of primers is labeled. Thelabel may, for example, be a radioactive label, such as ³²P, ³³P or ³⁵S.In a preferred embodiment of the invention, the label is anon-radioactive label, for example, digoxigenin, biotin and fluorescencedye or a dye.

In another preferred embodiment said primers are selected from the groupconsisting of SEQ ID NOs: 52 to 111.

In yet another embodiment, the present invention relates to acomposition comprising a nucleic acid molecule, a vector, a polypeptide,an antibody, an aptamer and/or a primer or pair of primers of theinvention.

The term “composition”, as used in accordance with the presentinvention, relates to compositions which comprise at least one nucleicacid molecule, vector, polypeptide, an antibody and/or primer or pair ofprimers of this invention. It may, optionally, comprise furthermolecules capable of altering the characteristics of the component ofthe invention thereby, for example, suppressing, blocking, modulatingand/or activating their function which have neuroprotective, nootropic,antidepressive and/or cell-protective properties as will also bedescribed herein below. The composition may be in solid, liquid orgaseous form and may be, inter alia, in the form of (a) powder(s), (a)tablet(s), (a) solution(s) or (an) aerosol(s).

In a preferred embodiment the composition according to the invention isa diagnostic composition, optionally further comprising suitable meansfor detection. As described above, the present invention is based on thesurprising finding that mutations in the P2X7R protein are connectedwith affective disorders. Thus, this knowledge now allows to diagnoseaffective disorders in an easy way. The diagnostic composition comprisesat least one of the aforementioned compounds of the invention. Thediagnostic composition may be used, inter alia, for methods fordetermining the presence and/or expression of the nucleic acids and/orpolypeptides of the invention. This may be effected by detecting, e.g.,the presence of a corresponding gene in the genetic material of anindividual or the presence of the corresponding mRNA which comprisesisolation of DNA or RNA from a cell derived from said individual,contacting the DNA or RNA so obtained with a nucleic acid probe asdescribed above under hybridizing conditions, and detecting the presenceof mRNAs hybridized to the probe. Alternatively, the diagnosticcomposition may also be used for detecting the presence of a nucleicacid molecule of the invention by PCR. Furthermore, polypeptides of theinvention can be detected with methods known in the art, which comprise,inter alia, immunological methods, like, RIA, FIA, ELISA, FACS orWestern blotting.

Furthermore, the diagnostic composition of the invention may be useful,inter alia, in detecting the prevalence, the onset or the progress of adisease related to the expression of a polypeptide of the invention.Accordingly, the diagnostic composition of the invention may be used,inter alia, for assessing the prevalence, the onset and/or the diseasestatus of affective disorders, as defined herein above. It is alsocontemplated that the diagnostic composition of the invention may beuseful in discriminating (the) stage(s) of a disease.

The diagnostic composition optionally comprises suitable means fordetection. The nucleic acid molecule(s), vector(s), host(s),antibody(ies), aptamer(s), polypeptide(s) described above are, forexample, suitable for use in immunoassays in which they can be utilizedin liquid phase or bound to a solid phase carrier. Examples ofwell-known carriers include glass, polystyrene, polyvinyl ion,polypropylene, polyethylene, polycarbonate, dextran, nylon, amyloses,natural and modified celluloses, polyacrylamides, agaroses, andmagnetite. The nature of the carrier can be either soluble or insolublefor the purposes of the invention.

Solid phase carriers are known to those in the art and may comprisepolystyrene beads, latex beads, magnetic beads, colloid metal particles,glass and/or silicon chips and surfaces, nitrocellulose strips,membranes, sheets, duracytes and the walls of wells of a reaction tray,plastic tubes or other test tubes. Suitable methods of immobilizingnucleic acid molecule(s), vector(s), host(s), antibody(ies), aptamer(s),polypeptide(s), etc. on solid phases include but are not limited toionic, hydrophobic, covalent interactions or (chemical) crosslinking andthe like. Examples of immunoassays which can utilize said compounds ofthe invention are competitive and non-competitive immunoassays in eithera direct or indirect format. Commonly used detection assays can compriseradioisotopic or non-radioisotopic methods. Examples of suchimmunoassays are the radioimmunoassay (RIA), the sandwich (immunometricassay) and the Northern or Southern blot assay. Furthermore, thesedetection methods comprise, inter alia, IRMA (Immune RadioimmunometricAssay), EIA (Enzyme Immuno Assay), ELISA (Enzyme Linked Immuno Assay),FIA (Fluorescent Immuno Assay), and CLIA (Chemioluminescent ImmuneAssay). Furthermore, the diagnostic compounds of the present inventionmay be are employed in techniques like FRET (Fluorescence ResonanceEnergy Transfer) assays.

Appropriate labels and methods for labeling are known to those ofordinary skill in the art. Examples of the types of labels which can beused in the present invention include inter alia, fluorochromes (likefluorescein, rhodamine, Texas Red, etc.), enzymes (like horse radishperoxidase, β-galactosidase, alkaline phosphatase), radioactive isotopes(like 32P, 33P, 35S or 125I), biotin, digoxygenin, colloidal metals,chemi- or bioluminescent compounds (like dioxetanes, luminol oracridiniums).

A variety of techniques are available for labeling biomolecules, arewell known to the person skilled in the art and are considered to bewithin the scope of the present invention and comprise, inter alia,covalent coupling of enzymes or biotinyl groups, phosphorylations,biotinylations, random priming, nick-translations, tailing (usingterminal transferases). Such techniques are, e.g., described in Tijssen,“Practice and theory of enzyme immunoassays”, Burden and von Knippenburg(Eds), Volume 15 (1985); “Basic methods in molecular biology”, Davis LG, Dibmer M D, Battey Elsevier (1990); Mayer, (Eds) “Immunochemicalmethods in cell and molecular biology” Academic Press, London (1987); orin the series “Methods in Enzymology”, Academic Press, Inc.

Detection methods comprise, but are not limited to, autoradiography,fluorescence microscopy, direct and indirect enzymatic reactions, etc.

Said diagnostic composition may be used for methods for detecting thepresence and/or abundance of a nucleic acid molecule of the invention ina biological and/or medical sample and/or for detecting expression ofsuch a nucleic acid molecule (e.g. by determining the mRNA or theexpressed polypeptide). Furthermore, said diagnostic composition mayalso be used in methods of the present invention, inter alia, for thedetection of specific antagonists or agonists for P2X7R ATP-gated ionchannels (see herein below).

In a further embodiment the present invention provides a method ofdiagnosing an affective disorder or a susceptibility to an affectivedisorder comprising the step of determining in a sample obtained from anindividual whether the P2X7R protein expressed in the cells of saidindividual is non-functional, shows an altered ATP-gating in comparisonto the wild-type P2X7R protein or is over- or under-expressed incomparison to the P2X7R protein level of an unaffected individual.

The term “over- or under-expressed in comparison to the P2X7R proteinlevel” in the context of the present invention means that the P2X7Rprotein level is higher or lower than the P2X7R level of an healthyindividual, i.e. an individual not affected with an affective disorder.The over-expression may result, e.g. from an increased amount of P2X7RmRNA caused by enhanced transcription rates due to increased activity ofthe RNA-polymerase II. The amount of mRNA may accordingly lead to anincreased translation and, thus, to an higher protein level of P2X7R. Itmay also be possible that a higher amount of P2X7R protein is caused byincreased stability of the protein. An under-expression of P2X7R proteinmay be caused by low transcription rates of the P2X7R gene and, thus,insufficient amounts of P2X7R mRNA give only rise to a low P2X7R proteinamount. Another reason may be that the P2X7R protein is unstable and,thus, is not present in amounts comparable to the wild-type proteinlevel.

The under- or over-expression of P2X7R protein may be determined bymethods well-known to the person skilled in the art. These include, butare not limited, to methods for determining the amount of mRNA or theamount and/or activity of the protein. Examples are Northern Blotanalysis or immuno based techniques, such as Western Blotting.

“Non-functional” means that the P2X7R protein has lost at least onefunctional property displayed by the wild-type P2X7R protein asdescribed herein above. Preferably, “non-functional” means that theP2X7R protein does no longer function as a channel. Non-functionalitymay, e.g., be caused by the fact that one allele occurring in anindividual codes for a P2X7R protein which leads to non-functionaldimers (dominant negative mutation). Whether a P2X7R protein in anindividual is functional or non-functional can be determined by themethods described herein above and in the examples.

The term “altered ATP-gating” means that the respective P2X7R proteinreacts in a different way to ATP than the wild-type P2X7R protein. Thiscan be determined as described in the appended examples or as describedhereinabove.

In the context of diagnosis, not only the activity of the P2X7R could beof diagnostic value but also the amount of expression. For example, if apolymorphism affects RNA stability or translation efficiency, this couldlead to lower expression of the P2X7 protein not only in the hippocampusbut also in the blood. Therefore, one could speculate that a loweramount of P2X7 detected by western blot in blood cells could be relatedto depression.

Another aspect of the present invention is a method for diagnosing anaffective disorder or a susceptibility to an affective disordercomprising the step of determining in a sample obtained from anindividual whether the P2X7R gene sequence or encoded protein thereofcomprises a mutation in comparison to the wild-type P2X7R sequence.

A preferred embodiment of the present invention is a method, wherein amutation is a mutation in a P2X7R sequence as defined hereinabove and/ora nucleotide replacement or deletion selected from the following Table Cindicating in column “Region of P2X7R” the region of the P2X7R genomicnucleotide sequence in which the replacement or deletion occurs, incolumn “Nucleotide” of Table C the nucleotide which is replaced byanother nucleotide or the nucleotides which are deleted and in column“Position in wild-type” of Table C the corresponding position in thenucleotide sequence of the wild-type ATP-gated ion channel P2X7R asdepicted in SEQ ID NO: 1 TABLE C Region of Position in P2X7R Nucleotidewild-type 5′UTR T 362 5′UTR T 532 5′UTR A 1100 5′UTR A 1122 5′UTR C 11715′UTR T 1351 5′UTR G 1702 5′UTR T 1731 5′UTR C 1860 5′UTR C 2162 5′UTR C2238 5′UTR A 2373 5′UTR G 2569 5′UTR G 2702 intron 1 G 3166 intron 1 C24778 intron 1 C 24830 exon 2 T 24942 exon 3 C 26188 exon 3 A 26308 exon3 G 26422 intron 4 G 32394 intron 4 T 32434 exon 5 G 32493 exon 5 G32506 exon 5 C 32507 exon 5 C 32548 intron 5 A 32783 intron 5 T 35309intron 5 C 35374 intron 5 A 35378 exon 6 G 35438 exon 6 T 35454 intron 6T 35549 intron 6 G 35641 intron 6 A 35725 intron 6 T 36001 intron 6 A36064 intron 6 deletion of GTTT 36091 to 36094 intron 6 C 36108 intron 7C 36374 intron 7 G 36378 intron 7 T 36387 intron 7 G 36398 intron 7 C37439 intron 7 T 37513 exon 8 C 37604 exon 8 G 37605 exon 8 G 37623 exon8 C 37633 intron 9 C 47214 exon 11 G 47383 exon 11 C 47411 intron 11 T47563 intron 12 C 54307 intron 12 G 54308 exon 13 C 54399 exon 13 A54480 exon 13 C 54523 exon 13 deletion of 54562 to 54582CCCTGAGAGCCACAGGTGCCT exon 13 A 54588 exon 13 C 54664 exon 13 G 54703exon 13 A 54804 exon 13 G 54834 exon 13 G 54847 3′UTR G 54925 3′UTR C55169 3′UTR A 55170 3′UTR A 55171 3′UTR C 55917

As indicated hereinabove, if the respective nucleotide which is replacedby another nucleotide is a purine base, it is preferred to be replacedby another purine base. If it is a pyrimidine base, it is preferred tobe replaced by another pyrimidine base. It is also preferred that apurine base is replaced by a pyrimidine base and that a pyrimidine baseis replaced by a purine base. Most preferably, the nucleotides indicatedin Table C are replaced by the nucleotides indicated at the respectiveposition in Table 12 hereinbelow (see Example 3).

In a preferred embodiment the present invention relates to diagnosticcomposition designed for use in a method in which the occurrence of themutation in the ATP-gated ion channel P2X7R gene is determined by PCR,immunological methods and/or electrophysiological methods as describedherein below and in the appended Examples. Additionally, it is possibleto determine the occurrence of a mutation in the ATP-gated ion channelP2X7R as described hereinabove.

In yet another aspect the present invention relates to the use of anucleic acid molecule, a vector, a polypeptide, an antibody, aptamerand/or a primer or pair of primers of the present invention for thepreparation of a diagnostic composition for the detection of anaffective disorder.

It is also envisaged that the present invention relates to methods ofdiagnosing an affective disorder of an individual comprising:

-   (a) isolating DNA from cells obtained from an individual;-   (b) determining all or part of the nucleotide composition of the    P2X7R gene; and-   (c) analyzing said nucleotide composition of P2X7R for the presence    of one or more polymorphism, mutation or allelic variation.

The term “gene” means a nucleotide sequence associated with theproduction of a protein, including promoter sequences, enhancersequences, intron sequences, exon sequences, coding regions, 5′untranslated region (5′UTR), 3′ untranslated region (3′UTR), and splicevariants.

In a preferred embodiment of the described method the individual is amammal and more preferably human. Moreover, the cells are preferablyderived from skin, blood, urine or cerebral spinal fluid.

The method of the present invention allows for the diagnosis of anaffective disorder according to the composition of a genetic markercorresponding to the P2X7R gene. As is demonstrated by the appendedexamples, polymorphisms in the P2X7R are genetically linked to patientssuffering from an affective disorder.

In accordance with this embodiment of the present invention, thediagnosis of an affective disorder can, e.g., be effected by isolatingcells from an individual, and isolating the genomic DNA of said cells.Such cells can be collected from body fluids, skin, hair, biopsies andother sources. Collection and analysis of cells from bodily fluids suchas blood, urine and cerebrospinal fluid is well known to the art; seefor example, Rodak, “Haematology: Clinical Principles & Applications”second ed., W B Saunders Co, 2002; Brunzel, “Fundamentals of Urine andBody Fluids Analysis”, W B Saunders Co, 1994; Herndon and Brumback(Ed.), “Cerebrospinal Fluid”, Kluwer Academic Pub., 1989. In addition,methods for DNA isolation are well described in the art; see, forexample, Sambrook et al., “Molecular Cloning: A Laboratory Manual”,3^(rd) edition, Cold Spring Harbor Laboratory, 2001.

Once DNA has been isolated, various oligonucleotide primers spanning theP2X7R locus may be designed in order to amplify the genetic material byPolymerase Chain Reaction (PCR). Conventional methods for designing,synthesizing, producing said oligonucleotide primers and performing PCRamplification may be found in standard textbooks, see, for exampleAgrawal (Ed.), “Protocols for Oligonucleotides and Analogs: Synthesisand Properties (Methods in Molecular Biology, 20)”, Humana Press, 1993;Innis et al. (Ed.), “PCR Applications: Protocols for FunctionalGenomics”, Academic Press, 1999; Chen and Janes (Ed.), “PCR CloningProtocols: From Molecular Cloning to Genetic”, 2^(nd) edition, HumanaPress, 2002. Primers for the detection of P2X7R polymorphisms are alsogiven in, but not limited to, SEQ ID NO: 52 to SEQ ID NO: 111. Once DNAhas been amplified, nucleotide structure can be analysed by sequencingmethods and compared to normal P2X7R DNA.

Sequencing may be performed manually by any molecular biologist ofordinary skills or by an automated sequencing apparatus. Theseprocedures are common in the art, see, for example, Adams et al. (Ed.),“Automated DNA Sequencing and Analysis”, Academic Press, 1994; Alphey,“DNA Sequencing: From Experimental Methods to Bioinformatics”, SpringerVerlag Publishing, 1997.

Detection and analysis of polymorphisms in P2X7R can also be performedusing amplification refractory mutation system (ARMSTM), amplificationrefractory mutation system linear extension (ALEXTM), single-strandconformation polymorphism (SSCP), heteroduplex analysis, PCR-SSCP,fluorescent SSCP in an automated DNA sequencer, denaturing gradient gelelectrophoresis, RNase protection assays, detection of mutations bysequence specific oligonucleotide hybridization, chemical cleavagemethods, enzyme mismatch cleavage methods, cleavage fragment lengthmethods, allele-specific oligonucleotide hybridization on DNA chips, andother such methods known in the art, see, for example Nollau et al,Clin. Chem. 43 (1997), 1114-1128; Burczak and Mardis (Ed.),“Polymorphism Detection & Analysis Techniques”, Eaton Pub Co, 2000;Cotton et al. (Ed.), “Mutation Detection: A Practical Approach”, IrlPress, 1998; Taylor (Ed.), “Laboratory Methods for the Detection ofMutations and Polymorphisms in DNA”, CRC Press, 1997.

The present invention also relates to a method of diagnosing anaffective disorder in an individual comprising:

-   (a) isolating RNA from cells obtained from an individual;-   (b) converting the RNA into cDNA;-   (c) determining all or part of the nucleotide composition of the    cDNA so obtained; and-   (c) analyzing said nucleotide composition for the presence of one or    more polymorphism(s) or allelic variation.

With respect to the preferred embodiments the same applies as alreadydescribed above.

Detection and analysis of polymorphisms in the P2X7R RNA can beperformed according to the methods described above.

The present invention also relates to a method for diagnosing anaffective disorder in an individual comprising:

-   (a) isolating RNA or proteins from cells obtained from an    individual;-   (b) determining the levels of P2X7R RNA or protein; and-   (c) comparing the levels of P2X7R RNA or protein with the    corresponding levels from a normal individual not afflicted with an    affective disorder.

With respect to the preferred embodiments the same applies as alreadydescribed above.

As is demonstrated by the appended examples, a relationship existsbetween the expression, or protein level of P2X7R and an affectivedisorder. This and other embodiments of the present invention willreadily occur to those of ordinary skill in the art in view of thedisclosure herein.

According to another aspect on the invention, there is provided apolynucleotide comprising at least 20 bases of the human P2X7R gene andcomprising a mutation or polymorphism selected from any of thefollowing: TABLE 1 Novel polymorphisms in the human P2X7R Region inProtein P2X7 Polymorphism Modification 5′UTR 362 T-C 5′UTR 532 T-G 5′UTR1100 A-G 5′UTR 1122 A-G 5′UTR 1171 C-G 5′UTR 1702 G-A Intron01 3166 G-CIntron01 24778 C-T Intron01 24830 6C-T Exon03 26188 C-T Arg117TrpIntron03 26308 A-G Intron03 26422 G-A Intron04 32394 G-A Intron04 32434T-C Exon05 32493 G-A Gly150Arg Exon05 32548 C-T Silent Cys168 Intron0532783 A-C Exon06 35438 G-A Glu186Lys Exon06 35454 T-C Leu191Pro Intron0635641 G-C Intron06 35725 A-C Intron06 36001 T-G Intron07 36378 G-AIntron07 36387 T-A Intron07 36398 G-C Exon08 37604 C-T Arg270Cys Exon0837633 C-T Silent Asp279 Intron09 47214 C-T Intron11 47563 T-C Intron1254307 C-T Intron12 54308 G-A Exon 13 54562-54582 deletion of deletion7aa of CCCTGAGA 488 to 494 GCCACAGGTGCCT (PESHRCL) Exon13 54804 A-TIle568Asn Exon13 54834 G-A Arg578Gln 3′UTR 55169 C-A 3′UTR 55170 A-C3′UTR 55171 A-C 3′UTR 55917 C-T 3′UTR 54925 G-A

The polymorphism describes the position and the variation observed. Theposition and numbering of the polymorphism corresponds to the humanP2X7R gene as defined in SEQ ID No 1. Primers used for SNP amplificationand sequencing are shown in Table 1 a and listed in SEQ ID NO: 52 to SEQID NO: 111. TABLE 1a Primer sequences for SNP amplification andsequencing Primer Name Orientation Sequence Begin End P2RX7_01.for Sensecgtaggacttggcgcttct 2785 2803 P2RX7_01.rev Antisensegagcacgtctcagattcgaaa 3224 3244 P2RX7_02.for Senseccatgaggcaggtatgactattc 24665 24687 P2RX7_02.rev Antisensectcctggatctcacccagtt 25168 25187 P2RX7_03.for Sensectcgtccagctttgatattaagc 25966 25988 P2RX7_03.rev Antisenseggtccctagtgctagaaccaga 26426 26447 P2RX7_04.for Sense attcatccgtcagtggcc30794 30811 P2RX7_04.rev Antisense gccatgtgaattttctaccgat 31277 31298P2RX7_05.for Sense ttcgttgtggttaggatggg 32314 32333 P2RX7_05.revAntisense caaggatgctcagggtagtagc 32805 32826 P2RX7_06.for Sensecactaggtttgctgtatccatttct 35277 35301 P2RX7_06.rev Antisensegcaactgtgtgagagcttgg 35731 35750 P2RX7_07.for Sense tcaaccctggtccagtgtg35950 35968 P2RX7_07.rev Antisense caccaagtagctctcactcataagg 36424 36448P2RX7_08.for Sense caataacacttgtgcgagttaggt 37380 37403 P2RX7_08.revAntisense catcttgttgccttggaaacc 37750 37770 P2RX7_09.for Sensegtgagtggtaatcctgctactgc 45321 45343 P2RX7_09.rev Antisenseaggcccactcctgtactcg 45743 45761 P2RX7_10_11.for Senseccaagtcacagcatgaggc 47119 47137 P2RX7_10_11.rev Antisenseacccagcgacgtatccac 47632 47649 P2RX7_12.for Sense aagcatggggttccatttc50252 50268 P2RX7_12.rev Antisense gcataaaagggactcctgctagta 50691 50714P2RX7_13a.for Sense gcttacagaacacatgcatgg 54232 54252 P2RX7_13a.revAntisense gcacctgtaggcacagtgc 54739 54757 P2RX7_13b.for Senseatcaccacctcagagctgttc 54620 54640 P2RX7_13b.rev Antisensegttaacatggctactgcagcc 55203 55223 P2XR7_13d.for Sensegcttagaaaggaggcgactcc 54484 54504 P2XR7_Pro13.for Sensettgtgacatttgcaaggctgcc 2617 2638 P2XR7_Pro7.rev Antisensetctgaagctctgctcctgag 1955 1974 P2XR7_Pro8.rev Antisensectcaccttctggcttccagt 1611 1630 P2XR7_Pro9.for Sense cttaccactcccaggactaa1496 1515 P2XR7_Pro10.for Sense gtctgcctgttcactgccat 1149 1168P2XR7_Pro1.for Sense cagagaccttcagaaacttcg 1841 1861 P2XR7_Pro2.revAntisense agatcaccagggacacagtg 2261 2280 P2XR7_Pro3.for Sensectcaactccactttcctcgg 2133 2152 P2XR7_Pro4.rev Antisensecctttcacttttttggtctcatg 2655 2677 P2XR7_Pro5.for Sensegggagaattctgaaaatgccc 2691 2711 P2XR7_Pro6.rev Antisenseggaccagagctctactcttc 2951 2970 P2XR7_Pro11.for Senseaggtcatagatcgacctgcc 2296 2315 P2XR7_Pro12.rev Antisenseaagaagcgccaagtcctacg 2785 2804 P2XR7_Pro14.for Sensegcaatccagactgaagttgac 2051 2071 P2XR7_Pro15.rev Antisenseactctggtctgcagttggtg 2428 2447 P2XR7_Pro21.for Sensecctttaaaatcagagaccttcaga 1831 1854 P2XR7_Pro22.for Sensegcccatcctctgaacaccat 2708 2727 P2XR7_3UTR10.for Sensecccttggaactcttgctatcg 55804 55824 P2XR7_3UTR1.for Senseggcagtacagtggcttcaaga 54858 54878 P2XR7_3UTR2.rev Antisensegtgggacagtttgctgtgcct 55150 55170 P2XR7_3UTR3.for Sensegagtccttaccaatagcagg 55183 55202 P2XR7_3UTR4.rev Antisensegtcaaagaatttgtggccacc 55643 55663 P2XR7_3UTR5.for Sensecatgaactgtcttttaatgtgtaaag 55515 55540 P2XR7_3UTR6.rev Antisensegagatacggtttcaccatgttg 55955 55976 P2XR7_3UTR7.for Senseaattagctgggcatggtgcg 55992 56011 P2XR7_3UTR8.rev Antisensettgagatggagtctcgctctg 56122 56140 P2XR7_3UTR9.rev Antisensecactgtccacgtgactgctt 56208 56227 P2RX7_11.For Sensetcctacttcggtctggtaagagatt 47281 47305 P2RX7_11.Rev Antisensegggcctaattttcgtgcat 47591 47609 P2RX7_13G.For Senseaagaacctagaacctgagggctt 54333 54355 P2RX7_13G.Rev Antisensettgagatgggaggcagctt 54541 54559 P2RX7_13H.For Sense ttcggctcccaggacat54773 54789 P2RX7_13H.Rev Antisense cacagagctttgcaggtgaa 55248 55267

Another aspect of the present invention is in the form of a diagnostickit for affective disorders comprising a specific oligonucleotide probe,or primer corresponding to P2X7R polymorphisms. The diagnostic kit maycomprise appropriate packaging and instructions for the use in themethod of the invention. Said kit may further comprise appropriatebuffer, and enzymes such as reverse transcriptase, and thermostablepolymerases.

In a preferred embodiment of the invention, diagnosis can be performedon a mouse, rat or human. The invention is generally applied in vitro,e.g. using cells or other material obtained from an individual. However,it can also be applied on a living individual, or post mortem.

In accordance with the embodiments of the present invention, diagnosisof an affective disorder may be followed by prescription, oradministration of an antidepressant drug. Administration and dosage ofantidepressive drugs can vary between patients and are well know in themedical art, see, for example Benkert and Hippius, “Kompendium derPsychiatrischen Pharmakotherapie”, Springer Verlag Publishing, 2000;Albers, “Handbook of Psychiatric Drugs: 2001-2002 Edition”, CurrentClinical Strategies Publishing, 2000. Preferred examples include between5 mg and 80 mg per day, preferably 20 mg, fluoxetine; between 5 mg and50 mg per day, preferably 20 mg, paroxetine; between 5 mg and 200 mg perday, preferably 50 mg, sertraline; between 5 mg and 300 mg per day,preferably 100 mg, fluvoxamine; between 5 mg and 100 mg per day,preferably 30 mg, mirtazapine; between 4 mg and 50 mg, preferably 8 mg,reboxetine; between 5 mg and 600 mg per day, preferably 200 mg,nefazodone; between 450 mg and 1800 mg per day, preferably 900 mg,lithium carbonate.

The P2X7R protein is also useful for monitoring the efficacy and/ordosing of a drug or the likelihood of a patient to respond to a drug.Thus, in yet another embodiment the invention relates to a method for,monitoring the efficacy and/or dosing of a drug, e.g. an antidepressivedrug, and/or the likelihood of a patient to respond to said drug whichcomprises determining the level of expression and/or activity of theP2X7R protein in a patient before and after administration of therespective drug. As presented in the examples below, treatment with anantidepressive drug results in an upregulation in P2X7R activity. Inhumans, P2X7R activity can be monitored by Positron Emission Tomography(PET) or Single Photon Emission Computerised Tomography (SPECT) using aradiolabelled ligand tracer for P2X7R. Examples of P2X7R ligands can be,but are not limited to, ATP, an antagonist binding P2X7R, an agonistbinding P2X7R, or a small polynucleotide comprising at least 20 bases ofthe human P2X7R gene. A modulation of P2X7R activity, membranedistribution or expression levels would reflect the activity and potencyof the antidepressive drug. Methods and techniques required for PETanalysis are well known in the art, see, for example Paans and Vaalburg,Curr. Pharmac. Design 6 (2000), 1583-1591; van Waarde, Curr. Pharmac.Design. 6 (2000), 1593-1610; Paans et al, Methods 27 (2002), 195-207;Passchier et al., Methods 27 (2002), 278-286; Laruelle et al., Methods27 (2002), 287-299.

In accordance with the present invention by the term “sample” isintended any biological sample obtained from an individual, cell line,tissue culture, or other source containing polynucleotides orpolypeptides or portions thereof. As indicated, biological samplesinclude body fluids (such as blood, sera, plasma, urine, synovial fluidand spinal fluid) and tissue sources found to express thepolynucleotides of the present invention. Methods for obtaining tissuebiopsies and body fluids from mammals are well known in the art. Abiological sample which includes genomic DNA, mRNA or proteins ispreferred as a source.

As described herein above, mutations of the P2X7R encoding gene canoccur on DNA level or on mRNA level and may result in an alteredexpression of P2X7R or in the expression of P2X7R ATP-gated ion channelswhich show either an altered function or no function when compared tothe wild-type P2X7R ATP-gated ion channel as described herein. Thus,various methods on DNA level, RNA level or protein level exist fordetermining whether the ATP-gated ion channel P2X7R gene shows amutation as described herein above. Consequently, mRNA, cDNA, DNA andgenomic DNA are the preferred nucleic acid molecules to be used in thebelow mentioned methods. Also polypeptides or fragments thereof arepreferred if a mutation in the P2X7R ATP-gated ion channel protein asdescribed herein is to be determined.

Preferably, a point mutation leading to the replacement of an amino acidresidue at the positions as indicated in Table 1 of the correspondingwild-type P2X7R amino acid sequence depicted in SEQ ID NO: 3 or 4 byanother amino acid can be determined by PCR. Said PCR is followed by arestriction fragment length polymorphism (RFLP) analysis if due to thepoint mutation a recognition site for a restriction endonuclease isgenerated which is not present in the wild-type nucleotide sequence or arecognition site for a restriction enzyme is created which does notoccur in the wild-type P2X7R. More preferably said mutation can bedetermined by PCR using primers and conditions that allow only anamplification of the wild-type nucleotide sequence encoding thecorresponding wild-type amino acid at the respective position, but notof the nucleotide sequence of a nucleic acid molecule encoding adifferent amino acid residue at the corresponding position. It is evenmore preferred that PCR is performed to determine a mutation usingprimers and conditions that allow no amplification if the wild-typenucleotide sequence is present, but only if another amino acid residueis encoded at the respective position. Particularly preferred is amethod using PCR and primers under conditions that allow amplificationof a fragment comprising at least the nucleotide residues encoding theamino acid residue corresponding to positions of SEQ ID NO: 1.

Said PCR is followed by e.g., sequencing and/or single strandconformation analysis (SSCA). Said fragment is preferably of at least 25nucleotides in length, more preferred of at least 50 nucleotide inlength, even more preferred of at least 75 nucleotides in length,particularly preferred of at least 100 nucleotides in length, moreparticularly preferred of at least 200 nucleotides in length, also moreparticularly preferred at least 250 nucleotides in length, even moreparticularly preferred at least 300 nucleotides in length and mostparticularly preferred at least 600 nucleotides in length. Said primersare preferably of at least 12 nucleotides in length, more preferred ofat least 15 nucleotides in length, even more preferred of at least 18nucleotides in length and most preferred of at least 21 nucleotides inlength as depicted in SEQ ID NOs: 52 to 111. The temperature forannealing said primers is preferably at least 50° C., more preferred atleast 55° C. and most preferred at least 58° C. The temperature fordenaturation is preferably at least 95° C. for preferably at least 10sec, more preferably at least 20 sec, even more preferred at least 30sec and most preferred at least 60 sec. However, depending on the lengthand the G-C content of the nucleic acid sequence to be amplified thetemperature for denaturation may be shorter or longer. The temperaturefor extension of the annealed primers is preferably at least 10 sec,more preferably at least 20 sec, even more preferred at least 30 sec andmost preferred at least 60 sec. A PCR reaction comprising theaforementioned conditions is exemplified in the Examples herein below.The subsequent sequencing and/or SSCA is carried out as known in theart. Preferably, the PCR fragments are separated on a 10% polyacrylamidegel at 4° C. or also preferred at room temperature. PCR fragmentsshowing a SSCA band shift are amplified with the primers underconditions as mentioned above and are subsequently sequenced.Alternatively, it is also possible to directly sequence genomic DNA inorder to determine whether a mutation in the CLCN2 gene has occurred. Adirect genomic sequencing approach is, for example, demonstrated forbakers yeast in Horecka, Yeast 16 (2000), 967-970.

Preferably, a deletion is determined by using hybridization techniquesas known in the art. In particular, a primer is designed as mentionedherein above that is capable to only hybridize to wild-type genomic DNAas depicted in SEQ ID NO: 1 but not to a nucleotide sequence comprisinga deletion of a fragment between nucleotides 54562 and 54582 of SEQ IDNO: 1. Also preferred is the method of fluorescent in situ hybridization(FISH) for determining on whole chromosomes, in particular on chromosome12q23-q24 that said chromosome has the above mentioned deletion. Evenmore preferred is that a deletion of nucleotide residues as describedherein may be determined by using PCR, wherein one primer of a pair ofprimers is located within the region of genomic DNA comprising saiddeletion. Preferably, said deletion is between nucleotide positions54562 and 54582 as depicted in SEQ ID NO: 1. Thus, under the appropriateconditions no PCR fragment will result if the genomic DNA comprises saiddeletion. It is particularly preferred that PCR using primers which arelocated upstream or downstream of the deletion is performed to determinesaid deletion. Under appropriate conditions as mentioned herein above,both a fragment of genomic DNA of the wild-type nucleotide sequence asset forth in SEQ ID NO: 1 and a fragment of the nucleotide sequencecomprising a deletion of preferably the nucleotides between positions54562 and 54582 as depicted in SEQ ID NO: 1 will be amplified.

It is also possible to determine the above-described P2X7R mutations onthe protein level. Some of the mutations described above lead toshortened versions of the P2X7R protein. Thus, it is conceivable todetermine the occurrence of these mutations by determining the length ormolecular weight of the P2X7R protein expressed in an individual, e.g.by SDS PAGE.

It is also possible to determine the mutations of the P2X7R ATP-gatedchannel as described herein by using the antibodies of the presentinvention. Said antibodies specific for said mutations of P2X7R proteinswill be determined by assay techniques such as radioimmunoassays,competitive-binding assays, Western blot analysis and ELISA assay. Alsopreferred are classical immunohistological methods.

The finding, described in the present invention, that certain mutationsin the P2X7R encoding gene and/or the corresponding protein areconnected with affective disorder is indicative that the non- ordysfunction of the P2X7R protein is responsible for various forms ofaffective disorders. Thus, the finding of these mutations not onlyallows the diagnosis of affective disorders by determining whether theabove-described mutations occur in an individual. It also allows todevelop a treatment of affective disorders which has been diagnosed tobe the result of a mutation in the P2X7R encoding gene. Such a treatmentcan, e.g., comprise the introduction of a nucleic acid molecule encodinga non-functional or functional wild-type P2X7R protein thereby restoringin said individual the P2X7R activity or the activation or repression of(a) P2X7R gene(s) in vivo. The term “activation or repression” in thiscontext means that the expression of the gene is either enhanced(activation) or reduced (repression). An enhancement of expression can,e.g., be achieved by increasing the efficiency of transcriptioninitiation, for example, by using suitable compounds which have anactivating effect on transcription. Alternatively, an enhancement can beachieved by replacing the naturally occurring promoter by a moreefficient promoter.

A repression may be achieved by suppressing expression of the gene,e.g., by specifically suppressing transcription from the respectivepromoter by suitable compounds or by rendering the promoter lessefficient or non-functional.

In another embodiment the present invention also relates to apharmaceutical composition. In accordance with the present invention theterm “pharmaceutical composition” relates to a composition comprising anucleic acid molecule comprising a nucleotide sequence which encodes anATP-gated ion channel P2X7R and which is selected from the groupconsisting of:

-   (a) a nucleotide sequence encoding a polypeptide comprising the    amino acid sequence as depicted in SEQ ID NO: 3 or 4;-   (b) a nucleotide sequence comprising the nucleotide sequence as    depicted in SEQ ID NO: 1 or SEQ ID NO: 2;-   (c) a nucleotide sequence which hybridizes to the nucleotide    sequence of (a) or (b); and-   (d) a nucleotide sequence which is degenerated as a result of the    genetic code to the nucleotide sequence of (c).

Such pharmaceutical compositions comprise a therapeutically effectiveamount of a nucleic acid molecule encoding a functional P2X7R proteinand, optionally, a pharmaceutically acceptable carrier. Thepharmaceutical composition may be administered with a physiologicallyacceptable carrier to a patient, as described herein. In a specificembodiment, the term “pharmaceutically acceptable” means approved by aregulatory agency or other generally recognized pharmacopoeia for use inanimals, and more particularly in humans. The term “carrier” refers to adiluent, adjuvant, excipient, or vehicle with which the therapeutic isadministered.

Such pharmaceutical carriers can be sterile liquids, such as water andoils, including those of petroleum, animal, vegetable or syntheticorigin, such as peanut oil, soybean oil, mineral oil, sesame oil and thelike. Water is a preferred carrier when the pharmaceutical compositionis administered intravenously. Saline solutions and aqueous dextrose andglycerol solutions can also be employed as liquid carriers, particularlyfor injectable solutions. Suitable pharmaceutical excipients includestarch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk,silica gel, sodium stearate, glycerol monostearate, talc, sodium ion,dried skim milk, glycerol, propylene, glycol, water, ethanol and thelike. The composition, if desired, can also contain minor amounts ofwetting or emulsifying agents, or pH buffering agents. Thesecompositions can take the form of solutions, suspensions, emulsion,tablets, pills, capsules, powders, sustained-release formulations andthe like. The composition can be formulated as a suppository, withtraditional binders and carriers such as triglycerides. Oral formulationcan include standard carriers such as pharmaceutical grades of mannitol,lactose, starch, magnesium stearate, sodium saccharine, cellulose,magnesium carbonate, etc. Examples of suitable pharmaceutical carriersare described in “Remington's Pharmaceutical Sciences” by E. W. Martin.Such compositions will contain a therapeutically effective amount of theaforementioned compounds, preferably in purified form, together with asuitable amount of carrier so as to provide the form for properadministration to the patient. The formulation should suit the mode ofadministration.

In another preferred embodiment, the composition is formulated inaccordance with routine procedures as a pharmaceutical compositionadapted for intravenous administration to human beings. Typically,compositions for intravenous administration are solutions in sterileisotonic aqueous buffer. Where necessary, the composition may alsoinclude a solubilizing agent and a local anesthetic such as lignocaineto ease pain at the site of the injection. Generally, the ingredientsare supplied either separately or mixed together in unit dosage form,for example, as a dry lyophilised powder or water free concentrate in ahermetically sealed container such as an ampoule or sachette indicatingthe quantity of active agent. Where the composition is to beadministered by infusion, it can be dispensed with an infusion bottlecontaining sterile pharmaceutical grade water or saline. Where thecomposition is administered by injection, an ampoule of sterile waterfor injection or saline can be provided so that the ingredients may bemixed prior to administration.

The pharmaceutical composition of the invention can be formulated asneutral or salt forms. Pharmaceutically acceptable salts include thoseformed with anions such as those derived from hydrochloric, phosphoric,acetic, oxalic, tartaric acids, etc., and those formed with cations suchas those derived from sodium, potassium, ammonium, calcium, ferrichydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol,histidine, procaine, etc.

In vitro assays may optionally be employed to help identify optimaldosage ranges. The precise dose to be employed in the formulation willalso depend on the route of administration, and the seriousness of thedisease or disorder, and should be decided according to the judgment ofthe practitioner and each patient's circumstances. Effective doses maybe extrapolated from dose-response curves derived from in vitro oranimal model test systems. Preferably, the pharmaceutical composition isadministered directly or in combination with an adjuvant.

The pharmaceutical composition is preferably designed for theapplication in gene therapy. The technique of gene therapy has alreadybeen described above in connection with the nucleic acid molecules ofthe invention and all what has been said there also applies inconnection with the pharmaceutical composition. For example, the nucleicacid molecule in the pharmaceutical composition is preferably in a formwhich allows its introduction, expression and/or stable integration intocells of an individual to be treated.

For gene therapy, various viral vectors which can be utilized, forexample, adenovirus, herpes virus, vaccinia, or, preferably, an RNAvirus such as a retrovirus. Examples of retroviral vectors in which asingle foreign gene can be inserted include, but are not limited to:Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus(HaMuSV), murine mammary tumor virus (MuMTV), and Rous Sarcoma Virus(RSV). A number of additional retroviral vectors can also incorporatemultiple genes. All of these vectors can transfer or incorporate a genefor a selectable marker so that transduced cells can be identified andgenerated. By inserting a P2X7R sequence of interest encoding afunctional P2X7R protein into the viral vector, along with another genewhich encodes, for example, the ligand for a receptor on a specifictarget cell, for example, the vector is now target specific. Retroviralvectors can be made target specific by inserting, for example, apolynucleotide encoding a sugar, a glycolipid, or a protein. Those ofskill in the art will know of, or can readily ascertain without undueexperimentation, specific polynucleotide sequences which can be insertedinto the retroviral genome to allow target specific delivery of theretroviral vector containing the inserted polynucleotide sequence.

Since recombinant retroviruses are preferably defective, they requireassistance in order to produce infectious vector particles. Thisassistance can be provided, for example, by using helper cell lines thatcontain plasmids encoding all of the structural genes of the retrovirusunder the control of regulatory sequences within the LTR. These plasmidsare missing a nucleotide sequence which enables the packaging mechanismto recognize an RNA transcript for encapsidation. Helper cell lineswhich have deletions of the packaging signal include, but are notlimited to w2, PA317 and PA12, for example. These cell lines produceempty virions, since no genome is packaged. If a retroviral vector isintroduced into such cells in which the packaging signal is intact, butthe structural genes are replaced by other genes of interest, the vectorcan be packaged and vector virion produced. Alternatively, NIH 3T3 orother tissue culture cells can be directly transfected with plasmidsencoding the retroviral structural genes gag, pol and env, byconventional calcium phosphate transfection. These cells are thentransfected with the vector plasmid containing the genes of interest.The resulting cells release the retroviral vector into the culturemedium. Another targeted delivery system for P2X7R polynucleotides is acolloidal dispersion system. Colloidal dispersion systems includemacromolecule complexes, nanocapsules, microspheres, beads, andlipid-based systems including oil-in-water emulsions, micelles, mixedmicelles, and liposomes. The preferred colloidal system of thisinvention is a liposome. Liposomes are artificial membrane vesicleswhich are useful as delivery vehicles in vitro and in vivo. It has beenshown that large unilamellar vesicles (LUV), which range in size from0.2-4.0 pm can encapsulate a substantial percentage of an aqueous buffercontaining large macromolecules. RNA, DNA and intact virions can beencapsulated within the aqueous interior and be delivered to cells in abiologically active form (Fraley, et al., Trends Biochem. Sci., 6:77,1981). In addition to mammalian cells, liposomes have been used fordelivery of polynucleotides in plant, yeast and bacterial cells. Inorder for a liposome to be an efficient gene transfer vehicle, thefollowing characteristics should be present: (1) encapsulation of thegenes of interest at high efficiency while not compromising theirbiological activity; (2) preferential and substantial binding to atarget cell in comparison to non-target cells; (3) delivery of theaqueous contents of the vesicle to the target cell cytoplasm at highefficiency; and (4) accurate and effective expression of geneticinformation (Mannino, et al., Biotechniques, 6:682, 1988). Thecomposition of the liposome is usually a combination of phospholipids,particularly high-phase-transition-temperature phospholipids, usually incombination with steroids, especially cholesterol. Other phospholipidsor other lipids may also be used. The physical characteristics ofliposomes depend on pH, ionic strength, and the presence of divalentcations. Examples of lipids useful in liposome production includephosphatidyl compounds, such as phosphatidylglycerol,phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine,sphingolipids, cerebrosides, and gangliosides. Particularly useful arediacylphosphatidylglycerols, where the lipid moiety contains from 14-18carbon atoms, particularly from 16-18 carbon atoms, and is saturated.Illustrative phospholipids include egg phosphatidylcholine,dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine. Thetargeting of liposomes can be classified based on anatomical andmechanistic factors. Anatomical classification is based on the level ofselectivity, for example, organ-specific, cell-specific, andorganelle-specific. Mechanistic targeting can be distinguished basedupon whether it is passive or active. Passive targeting utilizes thenatural tendency of liposomes to distribute to cells of thereticulo-endothelial system (RES) in organs which contain sinusoidalcapillaries.

In another aspect the present invention relates to a method of treatingan affective disorder comprising administering a therapeuticallyeffective amount of the pharmaceutical composition comprising anucleotide sequence encoding a functional ATP-gated ion channel asdescribed herein above to a subject suffering from said disorder.

In the context of the present invention the term “subject” means anindividual in need of a treatment of an affective disorder. Preferably,the subject is a vertebrate, even more preferred a mammal, particularlypreferred a human.

The term “administered” means administration of a therapeuticallyeffective dose of the aforementioned nucleic acid molecule encoding afunctional P2X7R protein to an individual. By “therapeutically effectiveamount” is meant a dose that produces the effects for which it isadministered. The exact dose will depend on the purpose of thetreatment, and will be ascertainable by one skilled in the art usingknown techniques. As is known in the art and described above,adjustments for systemic versus localized delivery, age, body weight,general health, sex, diet, time of administration, drug interaction andthe severity of the condition may be necessary, and will beascertainable with routine experimentation by those skilled in the art.

The methods are applicable to both human therapy and veterinaryapplications. The compounds described herein having the desiredtherapeutic activity may be administered in a physiologically acceptablecarrier to a patient, as described herein. Depending upon the manner ofintroduction, the compounds may be formulated in a variety of ways asdiscussed below. The concentration of therapeutically active compound inthe formulation may vary from about 0.1-100 wt %. The agents maybeadministered alone or in combination with other treatments.

The administration of the pharmaceutical composition can be done in avariety of ways as discussed above, including, but not limited to,orally, subcutaneously, intravenously, intra-arterial, intranodal,intramedullary, intrathecal, intraventricular, intranasally,intrabronchial, transdermally, intranodally, intrarectally,intraperitoneally, intramuscularly, intrapulmonary, vaginally, rectally,or intraocularly. In some instances, for example, in the treatment ofwounds and inflammation, the candidate agents may be directly applied asa solution dry spray.

The attending physician and clinical factors will determine the dosageregimen. As is well known in the medical arts, dosages for any onepatient depends upon many factors, including the patient's size, bodysurface area, age, the particular compound to be administered, sex, timeand route of administration, general health, and other drugs beingadministered concurrently. A typical dose can be, for example, in therange of 0.001 to 1000 μg; however, doses below or above this exemplaryrange are envisioned, especially considering the aforementioned factors.

The dosages are preferably given once a week, however, duringprogression of the treatment the dosages can be given in much longertime intervals and in need can be given in much shorter time intervals,e.g., daily. In a preferred case the immune response is monitored usingherein described methods and further methods known to those skilled inthe art and dosages are optimized, e.g., in time, amount and/orcomposition. Dosages will vary but a preferred dosage for intravenousadministration of DNA is from approximately 10⁶ to 10¹² copies of theDNA molecule. If the regimen is a continuous infusion, it should also bein the range of 1 μg to 10 mg units per kilogram of body weight perminute, respectively. Progress can be monitored by periodic assessment.The pharmaceutical composition of the invention may be administeredlocally or systemically. Administration will preferably be parenterally,e.g., intravenously. Preparations for parenteral administration includesterile aqueous or non-aqueous solutions, suspensions, and emulsions.Examples of non-aqueous solvents are propylene glycol, polyethyleneglycol, vegetable oils such as olive oil, and injectable organic esterssuch as ethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium ion solution, Ringer'sdextrose, dextrose and sodium ion, lactated Ringer's, or fixed oils.Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives may also be present such as,for example, antimicrobials, anti-oxidants, chelating agents, and inertgases and the like.

It is also envisaged that the pharmaceutical compositions are employedin co-therapy approaches, i.e. in co-administration with othermedicaments or drugs, for example other drugs for preventing, treatingor ameliorating affective disorders.

Another aspect of the present invention is a pharmaceutical compositioncomprising a compound, the administration of which to cells leads to areduction or increase of the expression of a nucleic acid encoding anATP-gated ion channel P2X7R in the cells or comprising a nucleic acidmolecule the expression of which in cells or the administration of whichto cells leads to a reduction or increase of the expression of a nucleicacid encoding an ATP-gated ion channel P2X7R in the cells. Saidpharmaceutical composition may be useful for treating individuals havingan increased or reduced amount of the P2X7R protein or expression levelas described hereinabove. Preferably, said pharmaceutical compositionleads to in increase of the expression of a nucleic acid encoding anATP-gated ion channel P2X7R in the cells.

It is envisaged that the above-mentioned pharmaceutical composition, theadministration of which to cells leads to a reduction of the expressionof a nucleic acid encoding an ATP-gated ion channel P2X7R is anantisense nucleic acid, a ribozyme, a co-suppressive nucleic acid, iRNAor siRNA.

An siRNA approach is, for example, disclosed in Elbashir ((2001), Nature411, 494-498)). It is also envisaged in accordance with this inventionthat for example short hairpin RNAs (shRNAs) are employed in accordancewith this invention as pharmaceutical composition. The shRNA approachfor gene silencing is well known in the art and may comprise the use ofst (small temporal) RNAs; see, inter alia, Paddison (2002) Genes Dev.16, 948-958.

As mentioned above, approaches for gene silencing are known in the artand comprise “RNA”-approaches like RNAi or siRNA. Successful use of suchapproaches has been shown in Paddison (2002) loc. cit., Elbashir (2002)Methods 26, 199-213; Novina (2002) Mat. Med. Jun. 3, 2002; Donze (2002)Nucl. Acids Res. 30, e46; Paul (2002) Nat. Biotech 20, 505-508; Lee(2002) Nat. Biotech. 20, 500-505; Miyagashi (2002) Nat. Biotech. 20,497-500; Yu (2002) PNAS 99, 6047-6052 or Brummelkamp (2002), Science296, 550-553. These approaches may be vector-based, e.g. the pSUPERvector, or RNA polIII vectors may be employed as illustrated, interalia, in Yu (2002) loc. cit.; Miyagishi (2002) loc. cit. or Brummelkamp(2002) loc. cit.

A compound which leads to a reduction of the expression of the P2X7Rgene may, e.g., be a compound which acts on the regulatory region of thegene and thereby reduces the level of transcription. Such compounds canbe identified by methods as described herein below.

The invention also relates to the use of a nucleic acid moleculeencoding a functional P2X7R protein as described herein above inconnection with the pharmaceutical composition for the preparation of apharmaceutical composition for treating an affective disorder.

Furthermore, the present invention relates to a method of treating anaffective disorder comprising administering a therapeutically effectiveamount of the nucleic acid molecule according to the invention or atherapeutically effective amount of the corresponding encodedpolypeptide to a subject suffering from said disorder.

In another preferred embodiment the present invention relates to apharmaceutical composition comprising, inter alia, the polynucleotidesaccording to the present invention, i.e. polynucleotides havingmutations and/or deletions as described hereinabove. Such pharmaceuticalcompositions may, e.g., be useful for treating individuals having anincreased or decreased amount of the P2X7R protein or having a P2X7Rprotein showing an increased or decreased activity which can bedetermined as described hereinabove. Preferably, such a pharmceuticalcomposition may be useful for treating individuals having a decreasedamount of the P2X7R protein or having a P2X7R protein showing adecreased activity which can be determined as described hereinabove Itis envisaged that, e.g. a non-functional or preferably a hyperfunctionalP2X7R protein comprised by said pharmaceutical composition isincorporated in a P2X7R complex which naturally exists in cells asdescribed hereinabove. It is also envisaged that the above-describedtechniques for gene therapy can be used for treating an individual withthe nucleic acid molecules of the present invention, mutatis mutandis.

With respect to the possible modes of administration and preferredembodiments the same applies as has been set forth above.

Additionally, the present invention also envisages the use of thenucleic acid molecules, the vectors, the polypeptides, the antibodyand/or the aptamer according to the invention for the preparation of apharmaceutical composition for the treatment of an affective disorder.

A further aspect of the present invention is the use of a modulator ofP2X7R activity or expression for the preparation of a pharmaceuticalcomposition for treating an affective disorder. In the context of thepresent invention the term “modulator” means (a) compound(s), a complexof compounds, (a) substance(s) or complex of substances which canmodify, i.e. modulate the activity of P2X7R or the expression of P2X7Reither directly or indirectly. The modulation can, for example, occur atthe protein level. Particularly, the P2X7R protein may interfere withthe modulator such that it is either more active or less active. Themodulation can also occur on nucleic acid level. Namely, the gene istranscribed more frequently or less frequently giving rise to more orless protein. Modulation can also influence RNA or protein stability.Since it was surprisingly found that agonists of P2X7R improve thesymptoms of mice selected for anxiety and depressive behaviour, themodulator of P2X7R activity used for the preparation of a pharmaceuticalcomposition for treating an affective disorder is preferably an agonist.The term “agonist” means an agent or a compound that can interact with areceptor and initiate a physiological or a pharmacological responsecharacteristic of that receptor. Examples of P2X7R agonist include butare not restricted to ATP, ATP-4, BzATP(2′-3′-O-(4-Benzoylbenzoyl)adenosine 5′-triphosphate (C₂₄H₂₄N₅O₁₅P₃))and tenidap(5-chloro-2,3-dihydro-2-oxo-3-(2-thienylcarbonyl)-indole-1-carboxamide,i.e. C₁₅H₁₁ClN₂O₂S) or a derivative thereof. Particularly preferred,said agonist used to treat depression or anxiety is BzATP is asdemonstrated in Example 9 hereinbelow.

Although it was reported that activation of P2X7R could induce apoptosisand cell death in vitro (Di Virgillo et al., Cell Death Differ. 5(1988), 191-199; Virginio et al., J. Physiol. 519 (1999), 335-346), thepresent application demonstrates in Example 9 hereinbelow that treatmentof the brain of mice selected for anxiety and depressive behaviour withBzATP revealed no significant difference in the numbers of apoptoticcells between control mice and mice treated with BzATP. This resultindicates that activation of P2X7R did not result in cerebral cell deathin vivo which, thus, renders BzATP to be a candidate drug for treatmentof affective disorders.

It is furthermore envisaged that said modulator is selected from thegroup consisting of piperidine and piperazine derivatives, adamantanederivatives, substituted phenyl compounds, oxidized ATP,2-O-(4-benzoylbenzoyl)adenosine-5-triphosphate and3-O-(4-benzoylbenzoyl)adenosine-5-triphosphate as, for example,described in WO 99/29660, WO 99/29661; WO 99/296896; WO 00/61569; WO01/42194; WO 01/44170; WO 01/44213; WO 00/71529; WO 01/46200. Thefollowing compounds illustrate compounds which are also preferred to beused as modulators of P2X7R activity.

A compound of general formula:

where A is phenyl or a 5- or 6-membered heterocyclic ring containing oneor two heteroatoms selected from O, N or S; and optionally substitutedby C₁₋₆alkyl, halogen, nitro, amino, alkylamino, CF₃, SO₂Me, NHSO₂Me orcyano; B is C═O, NH or SO₂; X is C═O, CH(Me), O or (CH₂)p where p is 0or 1; Y is O, CH2, NH or S; Z is C═O or SO₂, provided that when Z isC═O, then Y is O, CH₂ or S; R is hydrogen or C₁₋₆alkyl; R¹ is hydrogen,halogen; R² is phenyl optionally substituted by CO₂H, CO₂alkyl, CONH₂ orR² is OH, NHR³, NHCH(R⁴)(CHR⁵)_(n)R⁶, NH—R⁷—R⁸, SO₂NHalkyl, NHCOalkyl,NHSO₂alkyl, morpholine, NR⁹R¹⁰, piperazine substituted by phenyl,alkoxyphenyl, pyridyl or fluorophenyl; n is 0, 1 or 2; R³ is hydrogen, abi- or tricyclic saturated ring system optionally containing a nitrogenatom, piperidinyl, alkylpyrollidine, ethynylcyclohexyl, a 5-memberedaromatic ring containing 2 or 3 heteroatoms, C₄₋₆ cycloalkyl optionallysubstituted by alkyl, cyano or hydroxy, or C₁₋₈ alkyl optionallycontaining an oxygen atom in the alkyl chain and being optionallysubstituted by one or more substituents selected from ethynyl, cyano,fluoro, dialkylamino, hydroxy, thioalkyl, CO₂R¹¹ or CONH₂; R⁴ ishydrogen or alkyl optionally substituted by hydroxy or alkoxy; R⁵ ishydrogen or hydroxy; R⁶ is CO₂R¹¹. NHCO₂R¹², CONH₂ or a 5 or 6-memberedsaturated ring containing an oxygen atom, a 5-membered heterocyclic ringcontaining one or two heteroatoms selected from O, N or S, or phenyloptionally substituted by one or more groups selected from alkyl,hydroxy, amino, alkoxy, or nitro; R⁶ is alkyl; R⁷ is a cyclopentanering; R⁸ is phenyl; R⁹ and R¹⁰ are independently hydrogen, benzyl,alkenyl, cycloalkyl, alkyl optionally substituted by hydroxy, alkoxy,cyano, dialkylamino, phenyl, pyridyl or CO₂R¹¹ or R⁹ and R¹⁰ togetherform a 5- to 7-membered saturated or partially saturated ring optionallycontaining a further heteroaton and optionally substituted by one ormore groups selected from alkyl (optionally containing an oxygen atom inthe chain and optionally substituted 14 by hydroxy), COalkyl, CO₂R¹¹,COR¹³R¹⁴, CHO or piperidine, R¹¹ is hydrogen or alkyl; R¹² is alkyl; andR¹³ and R¹⁴ are independently hydrogen or alkyl, is or apharmaceutically acceptable salt or solvate thereof.

A compound of general formula:

wherein A represents a group CH₂ or an oxygen atom; B represents ahydrogen or halogen atom; D represents a group CH₂, OCH₂, NHCH₂ orCH₂CH₂; R represents a phenyl, benzothiazolyl, indolyl, indazolyl,purinyl, pyridyl, pyrimidinyl or thiophenyl group, each of which may beoptionally substituted by one or more substituents independentlyselected from a halogen atom or a cyano, carboxyl, hydroxyl, nitro,halo-C₁-C₆-alkyl, —N(R¹)—C(═O)—R², —C(O)—NR³R⁴, —NR⁵R⁶,C₃-C8-cycloalkyl, 3- to 8-membered heterocyclyl, C₃-C₈-cycloalkyloxy,C₁-C₆-alkylcarbonyl, phenoxy, benzyl, C₁-C₆-alkylthio, phenylthio,C₁-C₆-alkoxycarbonyl, C₁-C₆-alkylsulphinyl or C₁-C₆-alkylsulphonylgroup, or a C₁-C₆-alkyl or C₁-C₆-alkoxy group optionally substituted byone or more substituents independently selected from a halogen atom oran amino, carboxyl, hydroxyl, C₁-C₆-alkoxy, (di)C₁-C₆-alkylamino,C₁-C₆-alkoxycarbonyl, imidazolyl, morpholinyl, piperidinyl orpyrrolidinyl group; R¹ represents a hydrogen atom or a C₁-C₆-alkyl orC₃-C₈-cycloalkyl group; R² represents a C₁-C₆-alkyl or C₃-C₈-cycloalkylgroup; and R³, R⁴, R⁵ and R⁶ each independently represent a hydrogenatom or a C₁-C₆-alkyl or C₃-C₈-Cycloalkyl group; with the provisos thatwhen A is CH₂, B is H and D is CH₂, then R does not represent a phenyl,ortho-carboxyphenyl, methylphenyl or para-phenoxyphenyl group, and thatwhen A is CH₂, D is CH₂ or CH₂CH₂ and R represents a substituted phenylgroup, the substituent or substituents present do not comprise, in anortho position, a C₁-C₆-alkoxy group substituted by an amino,(di)C₁-C₆-alkylamino, imdazolyl, morpholinyl, piperidinyl orpyrrolidinyl group; or a pharmaceutically acceptable salt or solvatethereof.

A compound of general formula:

wherein x represents 1 or 2; A represents a group CH₂ or an oxygen atom;B represents a hydrogen or halogen atom; R represents a phenyl, pyridyl,indolyl, indazolyl, pyrimidinyl or thiophenyl group, each of which maybe optionally substituted by one or more substituents independentlyselected from a halogen atom or an amino, cyano, carboxyl, hydroxyl,nitro, C₁-C₆-alkyl, halo-C₁-C₆-alkyl, —N(R¹)—C(═O)—R², —C(O)NR³R⁴,—NR⁵R⁶, C₃-C₈-cycloalkyl, 3- to 8-membered heterocyclyl,C₃-C₈-cycloalkyloxy, C₁-C₆-alkylcarbonyl, C₁-C₆-alkoxycarbonyl,C₁-C₆-alkylsulphinyl or C₁-C₆-alkylsulphonyl group, or a C₁-C₆-alkoxy,C₁-C₆-alkylamino, phenoxy, benzyl, C₁-C₆-alkylthio or phenylthio groupoptionally substituted by one or more substituents independentlyselected from a 15 halogen atom or an amino, cyano, carboxyl, hydroxyl,nitro, 1-pyrrolidinyl, 1-piperidinyl, C₁-C₆-alkyl, C₁-C₆-alkoxy,(di)C₁-C₆-alkylamino, halo-C₁-C₆-alkyl, C₁-C₆-alkoxycarbonyl or one ofthe following groups:

R¹ represents a hydrogen atom or a C₁-C₆-alkyl or C₃-C₈-cycloalkylgroup; R² represents a C₁-C₆-alkyl or C₃-C₈-cycloalkyl group; R³ and R⁴each independently represent a hydrogen atom or a C₁-C₆-alkyl orC₃-C₈-cycloalkyl group; R⁵ represents a-hydrogen atom or a C₁-C₆-alkylor C₃-C₈-cycloalkyl group; R⁶ represents a C₃-C₈-cycloalkyl group and,additionally, a C₁-C₆-alkyl group when R⁵ is not a hydrogen atom; R⁷represents a hydrogen atom or a C₁-C₆-alkyl or C₃-C₈-cycloalkyl group;R⁸ represents a C₁-C₆-alkyl or C₃-C₈-cycloalkyl group; R⁹ represents ahydrogen atom or a hydroxyl group; and R¹⁰ represents a hydrogen atom ora phenyl or imidazolyl group; with the provisos that R does notrepresent an unsubstituted pyridyl group when A represents a group CH₂and B represents a hydrogen atom, and that when R represents asubstituted phenyl, indolyl or indazolyl group, the substituent orsubstituents present do not comprise an amido, carboxyl,(di)C₁-C₆-alkylamido or C₁-C₆-alkoxycarbonyl group in an ortho position;or a pharmaceutically acceptable salt or solvate thereof.

A compound of general formula:

wherein D represents CH₂ or CH₂CH₂; E represents C(O)NH or NHC(O); R¹and R² each independently represent a hydrogen or halogen atom, or anamino, nitro, C₁-C₆-alkyl or trifluoromethyl group; R³ represents agroup of formula:

X represents an oxygen or sulphur atom or a group NH, SO or SO₂; Yrepresents an oxygen or sulphur atom or a group NR¹¹, SO or SO₂; Zrepresents a group —OH, —SH, —CO₂H, C₁-C₆-alkoxy, C₁-C₆-alkylthio,C₁-C₆-alkylsulphinyl, C₁-C₆-alkylsulphonyl, —NR⁶R⁷, —C(O)NR⁸R⁹,imidazolyl, 1-methylimidazolyl, —N(R¹⁰)C(O)—C₁-C₆-alkyl,C₁-C₆-alkylcarbonyloxy, C₁-C₆-alkoxycarbonyloxy, —OC(O)NR¹²R¹³,—OCH₂OC(O)R¹⁴OCHOC(O)OR¹⁵ or —OC(O)OCH)OR¹⁶, R⁴ represents a C₂-C₆-alkylgroup; R⁵ represents a C₁-C₆-alkyl group; R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹², andR¹³ each independently represent a hydrogen atom, or a C₁-C₆-alkyl groupoptionally substituted by at least one hydroxyl group; R¹¹ represents ahydrogen atom, or a C₁-C₆-alkyl group optionally substituted by at leastone substituent independently selected from hydroxyl and C₁-C₆-alkoxy;and R¹⁴, R¹⁵ and R¹⁶ each independently represent a C₁-C₆-alkylgroup;with the provisos that (i) when E represents NEC(O), X represents O, Sor NH and Y represents O, then Z represents —NR⁶R⁷ where R⁶ represents ahydrogen atom and R⁷ represents either a hydrogen atom or a C₁-C₆-alkylgroup substituted by at least one hydroxyl group, and (ii) when Erepresents NHC(O), X represents O, S or NH, Y represents NH, and R⁵represents CH₂CH₂, then Z is not —OH or imidazolyl; or apharmaceutically acceptable salt or solvate thereof.

A compound of general formula:

wherein D represents CH₂ or CH₂CH₂; E represents C(O)NH or NHC(O); R¹and R² each independently represent hydrogen, halogen, amino, nitro,C₁-C₆-alkyl or trifluoromethyl, but R¹ and R² may not bothsimultaneously represent hydrogen; R³ represents a group of formula

R⁴ represents a C₁-C₆-alkyl group; X represents an oxygen or sulphuratom or a group NR¹³, SO or SO₂; R⁵ represents hydrogen, or R⁵represents C₁-C₆-alkyl or C₂-C₆-alkenyl, each of which may be optionallysubstituted by at least one substituent selected from halogen, hydroxyl,(di)C₁-C₆-alkylamino, —Y—R⁶,

and a 5- or 6-membered heteroaromatic ring comprising from 1 to 4heteroatoms independently selected from nitrogen, oxygen and sulphurwhich heteroaromatic ring may itself be optionally substituted by atleast one substituent selected from halogen, hydroxyl and C₁-C₆-alkyl; Yrepresents an oxygen or sulphur atom or a group NH, SO or SO₂; R⁶represents a group —R⁷Z where R⁷ represents a C₂-C₆-alkyl group and Zrepresents an —OH, —CO₂H.—NR⁸R⁹, —C(O)NR¹⁰R¹¹ or —N(R¹²)C(O)—C₁-C₆-alkylgroup, and, in the case where Y represents an oxygen or sulphur atom ora group NH, R⁶ additionally represents hydrogen, C₁-C₆-alkyl,C₁-C₆-alkylcarbonyl, C₁-C₆-alkoxycarbonyl, —C(O)NR¹⁴R¹⁵, —CH2OC(O)R¹⁶,—CH2OC(O)OR¹⁷ or —C(O)OCH2OR¹⁸; R⁸, R⁹, R¹⁰, R¹¹ and R¹² eachindependently represent a hydrogen atom or a C₁-C₆-alkyl group; R¹³represents hydrogen, C₃-C₈-cycloalkyl, C₃-C₈-cycloalkylmethyl, or R¹³represents a C₁-C₆-alkyl group optionally substituted by at least onesubstituent selected from hydroxyl to and C₁-C₆-alkoxy; and R¹⁴, R¹⁵,R¹⁶, R¹⁷, and R¹⁸ each independently represent a C₁-C₆-alkyl group; withthe proviso that when E is C(O)NH, X is O, NH or N(C₁-C₆-alkyl), then R⁵is other than a hydrogen atom or an unsubstituted C₁-C₆-alkyl group; ora pharmaceutically acceptable salt or solvate thereof.

A compound of general formula:

wherein m represents 1, 2 or 3; each R¹ independently represents ahydrogen or halogen atom; A represents C(O)NH or NHC(O); Ar represents agroup

X represents a bond, an oxygen atom or a group CO, (CH₂)₁₋₆, CH═,(CH₂)₁₋₆O, O(CH₂)₁₋₆, O(CH₂)₂₋₆O, O(CH₂)₂₋₃O(CH₂)₁₋₃, CR′(OH),(CH₂)₁₋₃O(CH₂)₁₋₃, (CH₂)₁₋₃O(CH₂)₂₋₃O, NR⁵, (CH₂)₁₋₆NR⁵, NR⁵(CH₂)₁₋₆,(CH₂)₁₋₃NR⁵(CH₂)₁₋₃, O(CH₂)₂₋₆NR⁵, O(CH₂)₂₋₃NR⁵(CH₂)₁₋₃,(CH₂)₁₋₃NR⁵(CH₂)₂₋₃O, NR⁵(CH₂)₂₋₆O, NR⁵(CH₂)₂₋₃O(CH₂)₁₋₃, CONR⁵, NR⁵CO,S(O)_(n), S(O)_(n)CH₂, CH₂S(O)_(n), SO₂NR⁵ or NR⁵SO₂; n is 0, 1 or 2; R¹represents a hydrogen atom or a C₁-C₆-alkyl group; one of R² and R³represents a halogen, cyano, nitro, amino, hydroxyl, or a group selectedfrom (i) C₁-C₆-alkyl optionally substituted by at least oneC₃-C₆-cycloalkyl, (ii) C₃-C₈-cycloalkyl, (iii) C₁-C₆-alkyloxy optionallysubstituted by at least one C₃-C₆-cycloalkyl, and (iv)C₃-C₈-cycloalkyloxy, each of these groups being optionally substitutedby one or more fluorine atoms, and the other of R² and R³ represents ahydrogen or halogen atom; either R⁴ represents a 3- to 9-memberedsaturated or unsaturated aliphatic heterocyclic ring system containingone or two nitrogen atoms and optionally an oxygen atom, theheterocyclic ring system being optionally substituted by one or moresubstituents independently selected from fluorine atoms, hydroxyl,carboxyl, cyano, C₁-C₆-alkyl, C₁-C₆-hydroxyalkyl, —NR⁶R⁷,—(CH₂)_(r)NR⁶R⁷ and CONR⁶R⁷, r is 1, 2, 3, 4, 5 or 6; R⁵ represents ahydrogen atom or a C₁-C₆-alkyl or C₃-C₈-cycloalkyl group; R⁶ and R⁷ eachindependently represent a hydrogen atom or a C₁-C₆-alkyl,C₂-C₆-hydroxyalkyl or C₃-C₈-cycloalkyl group, or R⁶ and R⁷ together withthe nitrogen atom to which they are attached form a 3- to 8-memberedsaturated heterocyclic ring; with the provisos that, (a) when Arepresents C(O)NH and R⁴ represents an unsubstituted 3- to 8-memberedsaturated aliphatic heterocyclic ring system containing one nitrogenatom, then X is other than a bond, and (b) when A represents C(O)NH andX represents a group (CH₂)₁₋₆ or O(CH₂)₁₋₆, then R⁴ does not representan unsubstituted imidazolyl, unsubstituted morpholinyl, unsubstitutedpiperidinyl or unsubstituted pyrrolidinyl group, and (c) when Arepresents NHC(O) and R⁴ represents an unsubstituted 3- to 8-memberedsaturated aliphatic heterocyclic ring system containing one nitrogenatom, then X is other than a bond, and (d) when A represents NHC(O) andX represents O(CH₂)₁₋₆, NH(CH₂)₁₋₆ or SCH₂, then R⁴ does not representan unsubstituted 1-piperidinyl or unsubstituted 1-pyrrolidinyl group,and (e) when A represents NHC(O) and X represents O(CH₂)₂₋₃NH(CH₂)₂,then R⁴ does not represent an imidazolyl group; or a pharmaceuticallyacceptable salt or solvate thereof.

A compound of general formula:

X represents a nitrogen atom or a group C(R⁵); Y represents an oxygen orsulphur atom or a group NR⁶; either R¹ and R² each independentlyrepresent a hydrogen atom or a C₁-C₄-alkyl group but do not bothsimultaneously represent a hydrogen atom, or R¹ and R² togetherrepresent a group —CH₂ZCH₂—; Z represents a bond, an oxygen or sulphuratom or a group CH₂ or NR⁷; m is 0 or 1; R³ represents a 5- to10-membered unsaturated ring system which may comprise from 1 to 4 ringheteroatoms independently selected from nitrogen, oxygen and sulphur,the ring system being optionally substituted by one or more substituentsindependently selected from halogen, nitro, cyano, NR⁸R⁹,C₁-C₄-alkyl-C(O)NH—, NHR¹²C(O)—, C₁-C₄-alkyl-SO₂—, C₁-C₄-alkyl-SO₂NH—,C₁-C₄-alkyl-NHSO₂—, C₁-C₄-alkoxy, and C₁-C₄-alkyl optionally substitutedby one or more fluorine atoms; R⁴ represents a phenyl or pyridinylgroup, each of which is substituted in an ortho position with asubstituent selected from halogen, C₁-C₄-alkoxy, C₁-C₄-alkylthio, andC₁-C₄-alkyl optionally substituted by one or more fluorine atoms, thephenyl or pyridinyl group being optionally further substituted by one ormore substituents independently selected from halogen, cyano, hydroxyl,C₁-C₄-alkylthio, C₁-C₄-alkyl-NH—, NHR¹³—C₁-C₄-alkyl-, C₁-C₄-alkyl-SO₂—,C₁-C₄-alkyl-SO₂NH—, C₁-C₄-alkyl-NHSO₂—, C₁-C₄-alkyl-C(O)NH—,C₁-C₄-alkyl-NHC(O)—, -D-G, C₁-C₄-alkoxy optionally substituted by—NR¹⁴R¹⁵ or by R¹⁶, and C₁-C₄-alkyl optionally substituted by one ormore fluorine atoms or by one or more hydroxyl groups, or R⁴ representsa 9- or 10-membered unsaturated bicyclic ring system which may comprisefrom 1 to 4 ring heteroatoms independently selected from nitrogen,oxygen and sulphur, the bicyclic ring system being optionallysubstituted by one or more substituents independently selected fromhalogen, oxo, C₁-C₄-alkyl, C₁-C₄-alkoxy, C₁-C₄-alkylthio and —NR¹⁰R¹¹; Drepresents an oxygen atom or a group (CH₂).sub.n or CH₂NH; n is 1, 2 or3; G represents a piperazinyl, morpholinyl or2,5-diazabicyclo[2.2.1]heptyl group, or G represents a piperidinyl groupoptionally substituted by amino; R⁵ represents a hydrogen atom, or ahydroxyl or C₁-C₄-alkoxy group; R⁶ represents a hydrogen atom, or acyano, nitro, hydroxyl, C₁-C₄-alkyl or C₁-C₄-alkoxy group; R⁷, R⁸ and R⁹each independently represent a hydrogen atom or a C₁-C₄-alkyl group; R¹⁰and R¹¹ each independently represent a hydrogen atom or a C₁-C₄-alkylgroup, or R¹⁰ and R¹¹ together with the nitrogen atom to which they areattached form a 5- or 6-membered saturated heterocyclic ring comprisingone or two ring nitrogen atoms; R¹² represents a hydrogen atom, or aC₁-C₄-alkyl group optionally substituted by amino; R¹³ represents ahydrogen atom, or a C₁-C₄-alkyl group optionally substituted byhydroxyl; R¹⁴ and R¹⁵ each independently represent a hydrogen atom or aC₁-C₄-alkyl group optionally substituted by hydroxyl, or R¹⁴ and R¹⁵together with the nitrogen atom to which they are attached form a 5- or6-membered saturated heterocyclic ring comprising one or two ringnitrogen atoms; and R¹⁶ represents a 1-(C₁-C₄-alkyl)-piperidinyl group;with the proviso that when m is 0, X is N and Y is O, then R⁴ does notrepresent 2-benzothiazolyl; or a pharmaceutically acceptable salt orsolvate thereof.

A compound of general formula:

wherein: each R¹ independently represents a hydrogen or halogen atom, ora trifluoromethyl, cyano, nitro, C₁-C₆-alkyl or C₁-C₆-alkoxy group; Trepresents an oxygen atom or a group NH; U represents an oxygen orsulphur atom or a group NH; Ar represents a group:

X represents a bond, an oxygen atom or a group CO, CH₂, CH₂O,O(CH2)_(m), CH₂OCH₂, NR⁵, CH₂NR⁵, NR⁵CH₂, CH₂NR⁵CH₂, CONR⁵, S(O)_(n) orSO₂NR⁵, m is 1. 2 or 3; 15 n is 0. 1 or 2; one of R² and R³ represents ahalogen, cyano, nitro, amino, hydroxyl, or a group selected fromC₁-C₆-alkyl optionally substituted by at least one C₃-C₆-Cycloalkyl,C₃-C₈-cycloalkyl, C₁-C₆-alkyloxy optionally substituted by at least oneC₃-C₆-cycloalkyl, C₃-C₈-cycloalkyloxy, S(O)_(p)C₁-C₆-alkyl orS(O)_(q)C₃-C₈-cycloalkyl, each of these groups being optionallysubstituted by one or more fluorine atoms, and, the other of R² and R³represents a hydrogen or halogen atom or a methyl group; p is 0, 1 or 2;q is 0, 1 or 2; R4 represents di(C₁₋₂alkyl)N(CH2)_(t) where t is 0, 1 or2 or imidazolyl, or R⁴ represents a 3- to 9-membered saturatedheterocyclic ring system containing one or two nitrogen atoms, theheterocyclic ring system being optionally substituted by one or moresubstituents independently selected from fluorine atoms, hydroxyl,C₁-C₆-alkyl, acetyl, hydroxyC₁-C₆-alkyl, —NR⁶R⁷, —(CH2)_(r)NR⁶R⁷,—CONR⁶R⁷ and pyrimidinyl, or R⁴ represents a 3- to 8-membered saturatedcarbocyclic ring system substituted by one or more substituentsindependently selected from —NR⁶R⁷, —(CH₂)_(r)NR⁶R⁷, —CONR⁶R⁷ the ringsystem being optionally further substituted by one or more substituentsindependently selected from fluorine atoms, hydroxyl and C₁-C₆-alkyl; ris 1, 2, 3, 4, 5 or 6; R⁵ represents a hydrogen atom or a C₁-C₆-alkyl orC₃-C₈-cycloalkyl group; and R⁶ and R⁷ each independently represent ahydrogen atom or a C₁-C₆-alkyl or C₃-C₈-cycloalkyl group, or R⁶ and R⁷together with the nitrogen atom to which they are attached form a 3- to8-membered saturated heterocyclic ring, provided that when R³ representsa cyano group, then X is other than a bond; or a pharmaceuticallyacceptable salt or solvate thereof.

A compound of general formula:

wherein X represents an oxygen or sulphur atom or a group NH, CH₂,CH₂CH₂ or OCH₂; Y represents a group CH₂ or C═O; R¹ represents a pyridylor pyrimidinyl group; R² represents a phenyl, pyridyl or pyrimidinylgroup, each of which may be optionally substituted by one or moresubstituents independently selected from a halogen atom or an amino,cyano, hydroxyl, nitro, C₁-C₆-alkyl, halo-C₁-C₆-alkyl, C₁-C₆-alkoxy,C₁-C₆-alkylthio, (di)C₁-C₆-alkylamino, C₁-C₆-alkylcarbonyl,C₁-C₆-alkoxycarbonyl, C₁-C₆-alkylsulphinyl, C₁-C₆-alkylsulphonyl,—NR³SO₂R⁴ or —SO₂NR⁵R⁶ group, or a group -Z-(CH₂)_(p)-Z-(CH₂)_(q)—Hwherein each Z independently represents a nitrogen or oxygen atom, p isan integer from 2 to 5 and q is 0 or an integer from 1 to 5; R³ and R⁴each independently represent a hydrogen atom or a C₁-C₆-alkyl group; andR⁵ and R⁶ each independently represent a hydrogen atom or a C₁-C₆-alkylgroup, or together with the nitrogen atom to which they are attachedform a pyrrolidinyl or piperidinyl group; or a pharmaceuticallyacceptable salt or solvate thereof.

An additional embodiment of the invention provides a method for treatingaffective disorders by administrating an agent modulating the activityof P2X7R, such as an antagonist of the P2X7R. The term “antagonist”means an agent or drug or a compound that opposes the physiologicaleffects of another. Examples of P2X7R antagonists include, but are notrestricted to, adamantane derivatives, isoquinolines and theirderivatives, substituted phenyl compounds, piperidine derivatives,piperazine derivatives. P2X7R antagonists are described in the art andinclude the compounds found in Chen et al., Bioconjugate Chem. 13(2002), 1100-1111; WO 99/29660; WO 99/29661; WO 99/296896; WO 00/61569;WO 01/42194; WO 01/44170; WO 01/44213; WO 00/71529; WO 01/46200. P2X7Ractivity can also be modulated by RNA-based interference mechanisms andmethods such as, but not limited to, small interference RNA (siRNA)molecules, and long double-stranded RNA (dsRNA).

Since it was unexpectedly found that agonists of P2X7R improve thesymptoms of mice selected for anxiety and depressive behaviour, thepresent invention relates in a further embodiment to a method fortreating affective disorders such as anxiety and depressive behaviour byadministrating an agent modulating the activity of P2X7R such as anagonist of the P2X7R. What is even more striking is the finding of thepresent application shown in Example 10 that antagonists of P2X7R haveno antidepressive effects although this is being taught by the priorart, for example, WO 03/042190, WO 03/042191, WO 03/049353 or US2004/0029841. WO 03/042190, WO 03/042191, WO 03/059353 or US2004/0029841 describe compositions of P2X7R antagonists and methods oftreating P2X7 mediated diseases by administering these compounds.However, while the prior art may make a link between P2X7R and, e.g.,treating depression, it is generally implied that antagonists of P2X7Rhave to be used in the treatment of, e.g., depression. Thus, the findingof the present invention that antagonists have no antidepressiveeffects, but rather agonists of P2X7R have an antidepressive effectcould not have been expected and is, thus, even more suprising. Thepresent application, therefore, provides, inter alia, the basis for thedevelopment of effective medicaments having therapeutic benefitsagainst, e.g., depression. In particular, said medicaments comprising anagonist of P2X7R are effective in treating affective disorders, inparticular for treating those disorders mentioned herein, and inparticular for treating depression. Examples of P2X7R agonists includebut are not restricted to ATP, ATP-4, and BzATP(2′-3′-O-(4-Benzoylbenzoyl)adenosine 5′-triphosphate (C₂₄H₂₄N₅O₁₅P₃)).Preferably, the P2X7R agonist to be used for treating affectivedisorders is BzATP. More preferably, BzATP is used to treat depressionor anxiety as demonstrated in Example 9 hereinbelow.

The present application provides another unexpected finding in that thechemical compound called tenidap or a derivative thereof also functionsas a modulator of P2X7R which can thus be used for treating affectivedisorders. So far, various medical applications are described fortenidap or a derivative thereof. However, the use of tenidap or aderivative thereof for treating affective disorders is neither known norsuggested in the prior art. Accordingly, the present application relatesto the use of tenidap or a derivative thereof or3-substituted-2-oxindole-1-carboxamides for the preparation of apharmaceutical composition for treating an affective disorder. Ofcourse, also a method of treatment of an affective disorder comprisingadministering a therapeutically effective amount of tenidap or aderivative thereof or 3-substituted-2-oxindole-1-carboxamides to asubject suffering from said disorder is envisaged. The composition oftenidap(5-chloro-2,3-dihydro-2-oxo-3-(2-thienylcarbonyl)-indole-1-carboxamide,i.e. C₁₅H₁₁ClN₂O₂S)) having the following structural formula

and other 3-substituted-2-oxindole-1-carboxamides and their use asanti-inflammatory and analgesic agents, and as inhibitors of both thecyclooxygenase (Cox) and lipoxygenase (5-LPO) enzymes was firstdisclosed in U.S. Pat. No. 4,556,672. Of course, various modificationsof, e.g., side groups or atoms which are well known in the art can bemade to the composition of tenidap.

Derivatives of tenidap or 3-substituted-2-oxindole derivatives aredescribed, for example, in U.S. Pat. No. 4,556,672; U.S. Pat. No.4,658,037; U.S. Pat. No. 4,721,712; U.S. Pat. No. 5,290,802; U.S. Pat.No. 5,118,703; U.S. Pat. No. 5,270,331; U.S. Pat. No. 5,298,522; U.S.Pat. No. 5,086,186, U.S. Pat. No. 5,449,788 and U.S. Pat. No. 5,795,902.Various processes for the synthesis of tenidap and other3-substituted-2-oxindole-1-carboxamides are well known in the art, seefor example U.S. Pat. No. 4,652,658; U.S. Pat. No. 4,665,194; U.S. Pat.No. 4,952,703; EP-B1 155 828, WO 90/04393, WO 94/07488, WO 94/17061, WO95/20574; WO 97/36895; van Deurzen et al., J. Mol. Catal. B-Enzym., 2(1996), 3342; Porcs-Makkay and Simig, Org. Process. Res. Dev., 4 (2000),10-16; Kumar et al., Org. Process. Res. Dev., 5 (2001), 61-64. Theanhydrous crystalline form of the sodium salt of tenidap is described inU.S. Pat. No. 5,036,099 and WO 88/05656. Injectable composition andpharmaceutical composition for rectal administration of tenidap arementioned in EP-B1 508 311 and EP-B1 508 310, respectively. It is alsodescribed in the prior art that tenidap or a derivative thereof can beadministered in combination with tetracycline (U.S. Pat. No. 5,308,839)or methotrexate (WO 96/35419) for the treatment of rheumatoid arthritis.Inhibition of the photodecomposition of tenidap and other3-substituted-2-oxindole-1-carboxamides is disclosed in WO 96/33701.

Further applications of tenidap or a derivative thereof and other3-substituted-2-oxindole-1-carboxamides have been described for theinhibition of interleukin-1 biosynthesis in mammals and for thetreatment interleukin-1 mediated disorders (U.S. Pat. No. 4,861,794);for the inhibition of elastase release from neutrophils (U.S. Pat. No.5,006,547); for the suppression of T-cell function in mammals and totreat T-cell mediated autoimmune disorders of the systemic or organspecific type (U.S. Pat. No. 4,853,409; Dolhain et al., Scand. J.Immunol. 42(1995), 686-693). Tenidap is also used for the treatment ofAlzheimer's disease (WO 96/31209; U.S. Pat. No. 5,545,656).

Tenidap or a derivative thereof or its pharmaceutically base salts havealso been shown to inhibit activation of collagenase, treat collagenasemediated disorders and diseases, and inhibit the activity ofmyeloperoxidase in mammals (U.S. Pat. No. 5,008,283). Tenidap can reducetotal serum cholesterol, LDL cholesterol and triglycerides (U.S. Pat.No. 5,122,534), and can be used for the treatment of ischemia inducedmyocardial injury and cytokine mediated myocardial injury (EP-B1 679396). However, none of the aforementioned documents discloses a use oftenidap or derivatives thereof or3-substituted-2-oxindole-1-carboxamides thereof for treating, forexample, affective disorders such as depression.

Sanz et al., Eur. J. Pharmacol. 355 (1998), 235-244 suggest that tenidapcan enhance the activity of the P2X7 receptor. It is suggested thattenidap may act by increasing ATP levels or improving the effect of ATPon P2X7. ATP is the natural ligand of P2X7R. Accordingly, tenidap or aderivative thereof is a modulator of P2X7R as is described herein sincea modulator is defined as either directly or indirectly modulating theactivity or expression of P2X7R. By making use of the teaching of thepresent invention that modulators of P2X7R are useful for treatingaffective disorders, it is envisaged that tenidap or a derivativethereof is used as a modulator of P2X7R activity for the preparation ofa pharmaceutical composition for the treatment of an affective disorder,examples of which are are described herein. The preparation ofpharmaceutical compositions, the modes of admininstration etc. aredescribed supra and infra and apply to the use of tenidap or aderivative thereof for the preparation of a pharmaceutical composition,mutatis mutandis. Moreover, also the embodiments relating to the uses ofand methods for treating affective disorders decribed herein apply tothe use of tenidap or a derivative thereof for treating affectivedisorders or the corresponding method of treatment, mutatis mutandis.

The present application moreover envisages that modulators of P2X7Ractivity can be used in any combinations thereof for treating anaffective disorder. For example, BzATP and tenidap or a derivativethereof or 3-substituted-2-oxindole-1-carboxamides may be used together,e.g., simultaneously or by successive administration for treating anaffective disorder.

In a preferred embodiment the pharmaceutical composition describedherein optionally comprises further molecules which have cell protectiveproperties capable of altering the characteristics of the components ofthe invention thereby, for example, modulating, preferably blockingpossible undesired, adverse or negative side effects of thesecomponents. One such possible undesired, adverse or negative side effectis the formation of pores in the cell membrane of treated cells whichultimatively leads to apoptosis. Accordingly, said further moleculesbelong to the class of beta-adrenergic receptor modulators includingagonists or antagonists having membrane-stabilizing properties.Beta-adrenergic receptor modulators including agonists and antagonistsare compounds which decrease or increase the positive chronotropic,positive inotropic, bronchodilator and vasodilator responses caused bybeta-adrenergic receptor agonists or antagonists. The magnitude of thisdecreased or increased response is proportional to the existingsympathetic tone and the concentration of beta-adrenergic receptorblocking agent which reaches the receptor sites. A beta-adrenergicreceptor modulator in the context of the present invention is thus anantagonist or agonist. The activity of a beta-adrenergic receptorantagonist or agonist can be determined as is well known in the art. Theactivity of beta-adrenergic receptors can be determined by measuring theaccumulation of cyclic adenosine mono-phosphate (cAMP) in Chinesehamster ovary (CHO) cells. CHO cells can be uniquely transfected withthe cDNA coding for the human beta1-, beta2-, or beta3-adrenergicreceptor under the control of the CMV promoter or any other suitablepromoter element. Transfection of the cells is performed using standardcell transfection methods, see for example, Joyner, “Gene Targeting: APractical Approach”, Oxford University Press, New York, 1993. Cellsoverexpressing one of the beta-adrenergic gene are then grown toconfluence in Ham's F12 media (Gibco BRL) containing 10% fetal bovineserum, 500 mg/ml Geneticin, 100 U/ml penicillin, 100 mg/ml streptomycinand 250 ng/ml fungizone according to the procedure described in AmericanType Culture Collection Catalogue of Cell Lines and Hybridomas, SeventhEdition, 1992, p. 36, ATCC CCL 61 CHO-K1. Beta-adrenergic modulatorcompounds can be prepared as 10 mM stock solutions in DMSO (0.1% DMSO,final concentration), diluted in Ham's F12 media and added to the cellsat 10⁻¹⁰ to 10⁻⁵ M along with 10⁻³ M isobutylmethylxanthine to inhibitphosphodiesterase activity. The media and cells are then incubated for 5minutes at 37° C. At the end of this period, the media is aspirated andthe cells lysed in 0.01 N HCl. The cellular content of cAMP can then bedetermined by radioimmunoassay (RIA) using a kit from New EnglandNuclear (Burlington, Mass.). There is a direct correlation between thecellular content of cAMP and the activation/inhibition of thebeta-adrenergic receptor. Other methods for determining the activity ofa beta-adrenergic receptors are well described in the art, see forexample, Vansal and Feller, J. Recept. Signal. Transduct. Res. 19 (1999)853-863; Durocher et al., Anal. Biochem. 284 (2000) 316-326.

Examples of compounds which fit the definition of a beta-adrenergicreceptor modulating agent include but are not limited to knownbeta-adrenergic receptor antagonist such as timolol, sotalol, esmolol,cateolol, propranolol, betaxolol, penbutolol, metoprolol, acebutolol,atenolol, metoprolol, pindolol, and bisoprolol, and their salts,hydrates, solvates and any crystal forms in which they may occur.Further examples of beta-adrenergic receptor blocking agents aredescribed in U.S. Pat. No. 5,776,930. Preferred examples ofbeta-adrenergic receptor antagonists are DL-propanolol, D-propanolol andlabetolol. DL-propanolol and labetolol are are beta-adrenergic receptorantagonists with membrane-stabilizing properties, while D-propanolol isan optical isomer with poor beta-adrenergic blocking activity. Theoptional addition of beta-adrenergic receptor antagonists or agonists tothe pharmaceutical composition of the present invention for treating anaffective disorder may be useful in the context of administering P2X7Ragonists. This is because the prior art suggests that the activation ofP2X7R by agonists may result in cell death by triggering the formationof pores within the cell membrane. However, as is demonstrated inExample 9 of the present application the pore-forming activity effectdescribed for P2X7R agonists by the prior art was only observed for veryfew cells. Recently, Alzola et al., Cell Signal. 13 (2001), 465-473 haveshown that concentrations of 10 to 300 μM DL-propanolol, D-propanolol orlabetolol can inhibit the pore-forming activity of P2X7R withoutaffecting the opening of the cation channel activity of P2X7R. From FR2768626 it is also known that beta-adrenergic modulators, e.g. agonistsare useful as apoptosis inhibiting agents. Accordingly, in a preferredembodiment of the present invention, beta-adrenergic receptor modulatorsincluding antagonists or agonists are administered in combination withP2X7R agonists for the treatment of affective disorders. Saidbeta-adrenergic receptor antagonists or agonists are preferablyadministered in a concentration of 10 to 300 μM.

Dosage, pharmaceutical preparation and delivery of P2X7R modulatingagent for use in accordance with the present invention may be formulatedin conventional manner according to methods found in the art, using oneor more physiological carriers or excipient, see, for example Ansel etal., “Pharmaceutical Dosage Forms and Drug Delivery Systems”, 7^(th)edition, Lippincott Williams & Wilkins Publishers, 1999. Thus, the P2X7Rmodulating agent and its physiologically acceptable salts and solvatesmay be formulated for administration by inhalation, insufflation (eitherthrough the mouth, or nose), oral, buccal, parenteral, or rectaladministration.

For oral administration, the pharmaceutical composition of the P2X7modulating agent may take the form of, for example, tablets or capsulesprepared by conventional means with pharmaceutical acceptable excipientssuch as binding agents (e.g., pregelatinised maize starch,polyvinylpyrrolidone, hydroxypropyl methylcellulose), fillers (e.g.,lactose, microcrystalline cellulose, calcium hydrogen phosphate),lubricants (e.g., magnesium stearate, talc, silica), disintegrants(e.g., potato starch, sodium starch glycolate), or wetting agents (e.g.,sodium lauryl sulphate). Liquid preparations for oral administration maytake the form of, for example, solutions, syrups, or suspensions, or maybe presented as a dry product for constitution with water or othersuitable vehicle before use. Such liquid preparation may be prepared byconventional means with pharmaceutically acceptable additives such assuspending agents (e.g., sorbitol, syrup, cellulose derivatives,hydrogenated edible fats), emulsifying agents (e.g., lecithin, acacia),non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol,fractionated vegetable oils), preservatives (e.g., methyl orpropyl-p-hydroxycarbonates, soric acids). The preparations may alsocontain buffer salts, flavouring, coloring and sweetening agents asdeemed appropriate. Preparations for oral administration may be suitablyformulated to give controlled release of the agent modulating P2X7Ractivity.

For administration by inhalation, the agent modulating P2X7R activityfor use according to the present invention is conveniently delivered inthe form of an aerosol spray presentation from a pressurised pack or anebulizer, with the use of a suitable propellant (e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas). In thecase of a pressurised aerosol, the dosage unit may be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof, for example, gelabne, for use in an inhaler or insufflator may beformulated containing a powder mix of the P2X7R activity modulatingagent and a suitable powder base such as lactose or starch.

An agent modulating P2X7R activity may be formulated for parenteraladministration by injection, for example, by bolus injection orcontinuous infusion. Site of injections include intra-venous,intra-peritoneal or sub-cutaneous. Formulations for injection may bepresented in units dosage form (e.g., in phial, in multi-dosecontainer), and with an added preservative. The agent modulating P2X7Ractivity may take such forms as suspensions, solutions or emulsions inoily or aqueous vehicles, and may contain formulatory agents such assuspending, stabilizing, or dispersing agents. Alternatively, the agentmay be in powder form for constitution with a suitable vehicle (e.g.,sterile pyrogen-free water) before use.

An agent modulating P2X7R activity may, if desired, be presented in apack, or dispenser device which may contain one or more unit dosageforms containing the said agent. The pack may for example comprise metalor plastic foil, such as blister pack. The pack or dispenser device maybe accompanied with instruction for administration.

In a more preferred embodiment the aforementioned methods or uses areenvisaged to treat affective disorders selected from the groupconsisting of major depression, generalized anxiety disorder and bipolardisorder.

In a particularly preferred embodiment said major depression is selectedfrom the group consisting of major depression, dysthymia, atypicaldepression, premenstrual dysphoric disorder and seasonal affectivedisorder.

In another particularly preferred embodiment said generalized anxietydisorder is selected from the group consisting of panic disorder,phobias, agoraphobia, social phobia, specific phobia,obsessive-compulsive disorder, post-traumatic stress disorder,separation anxiety disorder, mania, hypomania and cyclothymic disorder.

A still also particularly preferred embodiment is that said bipolardisorder is bipolar disorder type I or bipolar disorder type II.

Additionally, the present invention relates to a kit comprising thenucleic acid molecule, the vector, the host, the polypeptide, theantibody or the aptamer, the primer or pair of primers of the inventionor the molecule as identified or characterized in a method herein belowof the present invention.

Advantageously, the kit of the present invention further comprises,optionally (a) reaction buffer(s), storage solutions and/or remainingreagents or materials required for the conduct of scientific ordiagnostic assays or the like. Furthermore, parts of the kit of theinvention can be packaged individually in vials or bottles or incombination in containers or multicontainer units.

The kit of the present invention may be advantageously used, inter alia,for carrying out the method of producing a polypeptide of the invention,the method(s) of identification and/or characterization of moleculesspecifically interacting with P2X7R ATP-gated ion channels as describedherein below and/or it could be employed in a variety of applicationsreferred herein, e.g., as diagnostic kits, as research tools ortherapeutic tools. Additionally, the kit of the invention may containmeans for detection suitable for scientific, medical and/or diagnosticpurposes. The manufacture of the kits follows preferably standardprocedures which are known to the person skilled in the art.

Furthermore, the present invention relates to a method for identifyingcompounds or mixtures of compounds which are capable of specificallyinteracting with a polypeptide of the present invention, comprising thesteps of (a) contacting a polypeptide of the present invention with acandidate compound or mixture of compounds to be tested; and (b)determining whether said is capable of specifically interacting withsaid polypeptide. The polypeptide may be provided directly or byexpression of a corresponding nucleic acid molecule or vector of theinvention, e.g., in vitro or in a suitable host cell.

Additionally, the present invention relates to a method for thecharacterization of compounds which are capable of alteringcharacteristics of the polypeptides of the present invention, comprisingthe steps of (a) contacting a polypeptide of the invention with saidcompound; and (b) determining whether the compound alters acharacteristic of said polypeptide.

The term “altering characteristic of the polypeptide of the presentinvention” means that the functional characteristics to the polypeptidesof the present invention in comparison to functional characteristicswhich they had before being contacted with the compounds identified bythe above-described method: as described hereinabove are altered; i.e.changed.

Said identification and/or characterization of which are capable ofinteracting with or altering characteristics of the polypeptide of thisinvention, may be, inter alia, achieved by transfecting an appropriatehost with a nucleic acid molecule of invention. Said hosts comprise, butare not limited to, HEK 293 cells or are injected into frog oocytes,preferably a Xenopus oocyte for functional expression (Goldin, MethodsEnzymol. 207 (1992), 266). Expressed P2X7R ATP-gated channels can beexamined using standard two-electrode voltage clamp techniques (Stuhmer,Methods Enzymol. 207 (1992), 319; Kohler, Science 273 (1996), 1709).After expression of a P2X7R ATP-gated ion channel as defined herein,membrane currents may be deduced in the absence and/or presence of themolecule to be identified and/or characterized. Methods for thededuction of membrane currents are well known in the art and comprise,e.g., patch clamp methods as described in Hamill, Pfluger's Arch. 391(1981), 85-100 or two-electrode voltage clamp in oocytes, as describedin Methfessel, Pflügers Archive 407 (1986), 577-588. In accordance withthe present invention the term “interacting with the polypeptides of thepresent invention” means that the polypeptides of the present inventioninteract directly and/or indirectly with compounds identified by themethod described above. Furthermore, the present invention relates to amethod of screening for molecules which are capable of interacting withthe polypeptide of this invention, comprising the steps of (a)contacting a polypeptide of the invention with a molecule; and (b)measuring and/or detecting a response; and (c) comparing said responseto a standard response as measured in the absence of said candidatemolecule.

The present invention also relates to a method for identifying acompound which is capable of enhancing or reducing the expression of theP2X7R gene comprising the steps of contacting a cell which expresses theP2X7R gene from its natural promoter or a reporter gene driven by theP2X7R promoter and determining whether the expression of the gene isincreased or reduced when compared to conditions in which the compoundis not present.

Potential candidate molecules or candidate mixtures of molecules may be,inter alia, substances, compounds or compositions which are of chemicalor biological origin, which are naturally occurring and/or which aresynthetically, recombinantly and/or chemically produced or compounds orcompositions described hereinabove. Thus, candidate molecules may beproteins, protein-fragments, peptides, amino acids and/or derivativesthereof or other compounds, such as ions, which bind to and/or interactwith wild-type P2X7R ATP-gated ion channels. Such binding and/orinteracting candidate compounds may be found employing, inter alia,yeast two-hybrid systems or modified yeast two-hybrid systems asdescribed, for example in Fields, Nature 340 (1989), 245-246; Gyuris,Cell 75 (1993), 791-801; or Zervos, Cell 72 (1993), 223-232.

Furthermore, potential candidate molecules may be contacted with a cell,such as an oocyte or a HEK 293 cell, which expresses a polypeptide ofthe invention or with a membrane patch comprising a polypeptide of theinvention and a corresponding response (inter alia, a dose-responseresponse, a current-response, or single current channel response) may bemeasured in order to elucidate any effect said candidate moleculecauses.

Within the scope of the present invention are also methods foridentifying, characterizing and for screening of molecules which arecapable of interacting with the P2X7R ATP-gated ion channels accordingto the invention which comprise so-called high-throughput screeningmethods and similar approaches which are known in the art (Spencer,Biotechnol. Bioeng. 61 (1998), 61-67; Oldenburg, Annu. Rev. Med. Chem.33 (1998), 301-311) carried out using 96-well, 384-well, 1536-well (andother) commercially available plates. Further methods to be employed inaccordance with the present invention comprise, but are not limited to,homogenous fluorescence readouts in high-throughput screenings (asdescribed, inter alia, in Pope, Drug Discovery Today 4 (1999), 350-362).The method of the present invention for identification, characterizationand/or screening of molecules capable of interacting with P2X7RATP-gated ion channels can, inter alia, employ hosts as defined hereinwhich express the polypeptide of the present invention. Cell-basedassays, instrumentation for said assays and/or measurements arewell-known in the art and described, inter alia, in Gonzalez, DrugDiscovery Today 4 (1999), 431-439 or Ramm, Drug Discovery Today 4(1999), 401-410. It is also envisaged that the high through put screensdescribed herein are conducted by using, for example cRNA, i.e.synthetic RNA from a cDNA construct) that can be introduced in hostcells, such as Xenopus oocytes using routine methods in the art. As anexample, direct nucleic acid injection can be employed, such as theEppendorf microinjection system (Micromnipulator 5171 and Transjector5242). The injected/transformed cells can be analyzed for ion currentsabout 4 hours later using patch-clamp techniques which are commonlypracticed in the art.

Additionally, the present invention relates to a method for theproduction of a pharmaceutical composition comprising the steps of amethod of the invention for identifying, characterizing and/or screeningof molecules which are capable of interacting with and/or altering thecharacteristics of a P2X7R ATP-gated ion channel of the invention andfurther comprising a step, wherein a derivative of said identified,characterized and/or screened molecule is generated. Such a derivativemay be generated by, inter alia, peptidomimetics.

The invention furthermore relates to a method for the production of apharmaceutical composition comprising the steps of a method of theinvention for identifying, characterizing, screening and/or derivatizingof molecules which are capable of interacting with and/or altering thecharacteristics of a P2X7R ATP-gated ion channel and formulating themolecules identified, characterized, screened and/or derivatized inpharmaceutically acceptable form.

In a more preferred embodiment the present invention relates to a methodwherein said molecule(s) comprise(s) (a) neuroprotective, (a) nootropicand/or (a) antiepileptic molecule(s).

Yet another embodiment of the invention is the use of a P2X7Rpolypeptide, in particular those according to the present invention, toidentify biological, chemical, or pharmacological agents that can havean antidepressive effect. The term ‘agent’ refers to a chemical compoundor composition capable of inducing a desired therapeutic or prophylacticeffect when properly administered to a subject or cell. For example, thepresent invention allows the generation of cells expressing P2X7R forthe identification and characterization of agents which modulate ionicinflux and efflux. For example, HEK293 cells, or other cell lines (e.g.,HCN-1A, HCN-2, HIT-T15, RIN-m5F, betaTC3, PC12, HT22, SH-SY5Y, Neuro2Aor CA77), can be stably transfected with cDNA encoding the human P2X7Rand plated in 12, 96 and 384 well plates. Said cells are cultured inappropriate medium. Examples of such medium are well known in the art,see, for example Freshney, “Culture of Animal Cells: A Manual of BasicTechnique, 4th edition, Wiley-Liss Publishing, 2000.

Said cells can then be pre-incubated with said agents for 15 min priorto stimulation with 3 mM ATP for 10 minutes. Reactions are thenterminated by rapid aspiration of the extracellular medium in each well.The cells in each well are subsequently extracted overnight with 1 ml10% HNO3. Potassium (K+) content in the extracts can be determined byatomic absorbance spectrophotometry. Agent function is then measured bythe percent inhibition or stimulation of the K+ release triggered by 3mM ATP and compared to K+ release in the absence of the agents. P2X7Ractivity can also be monitored according to the movement of calcium(Ca2+; see Denver et al., Drug Discov. Today 7 (1998), 323-332; Gonzálezet al., Drug Discov. Today 9 (1999), 431-439; Helmchen and Waters, Eur.J. Pharmacol. 447 (2002), 119-129). Agents can also be verified in theabsence of ATP.

P2X7R activity can also be monitored according to secretion ofneurotransmitters such as glutamate and GABA. Neurotransmitter levels intreated cells can be quantified by suitable methods, e.g., Enzyme LinkedImmunoabsorbent Assay (ELISA), Radio Immuno Assay (RIA), HighPerformance Liquid Chromatography (HPLC). Using these methods, a largenumber of compounds can be screened for increase in neurotransmitter(for example, glutamate) secretion. The release of glutamate can bemeasured for example by Fluorometric glutamate release assays (e.g.,Amplex Red Glutamic Acid/Glutamate Oxidase Assay Kit, Molecular Probes)or High-Throughput ElectroPhysiology.

In a further aspect the present invention uses the P2X7R polypeptidesdisclosed herein or polypeptides of the present invention in a methodfor identifying compounds or agents having agonist activity to saidP2X7R polypeptides or to the polypeptides of the present invention.Agents and compounds are defined and described hereinabove andhereinbelow. In particular, cells that express the P2X7 gene arecontacted with candidate agents, molecules or compounds as describedhereinabove and either calcium influx or ethidium bromide entry ismeasured by methods known in the art, described hereinabove and inparticular described in Example 8 hereinbelow. The cells used in themethod for identifying agonists to P2X7R are preferably cells of ahippocampal cell line. Hippocampal cell lines are prepared by methodsknown in the art, for example, described in EP 0 773 287 or EP 0 773292. Non limiting examples of hippocampal cell lines are rat H19-7hippocampal cells (ATCC-2526) described in Eves et al. Proc. Natl. Acad.Sci. USA 89 (1992), 4373-4377, mouse HN9.10 hippocampal cells describedin Lee et al. J. Neurosci. 10 (1992), 1779-1787 or rat Hi5B hippocampalcells described in Renfranz et al., Cell 66 (1991), 713-729. Preferably,the hippocampal cells used in accordance with the aforementioned methodare cells of the HT-series (see Davis and Maher (1994), Brain Res. 652,169-173), Morimoto and Koshland (1990), Neuron 5, 875-880). It is alsopreferred that the hippocampal cells express the endogenous P2X7R gene.However, it is also envisaged that such cells may be geneticallymodified by introducing an exogenous P2X7R gene using methods commonlyknown in the art. More preferably, HT-22 cells are used for identifyingagonists to the P2X7R polypeptides described herein or to thepolypeptides of the present invention and HT-39 cells are used as anegative control as described in Example 8 hereinbelow.

In another embodiment, cells are transfected with nucleic acidconstructs encoding a reporter gene regulated by the P2X7R promoter (seeabove), an increase or decrease in the expression of the reporter genein response to biological or pharmaceutical agents can be analyzed usingmethods that detect levels or status of protein or mRNA present in thecorresponding cell or detect biological activities of the reporter gene.Suitable reporter molecules or labels, which may be used, includeradionucleotides, enzymes, fluorescent, chemiluminescent or chromogenicagents as well as substrates, co-factors, inhibitors, magneticparticles, and the like. Designing such drug screening assays are wellknown in the art; see Harvey ed., ‘Advances in drug discoverytechniques’, John Wiley and Sons, 1998; Vogel and Vogel eds., ‘Drugdiscovery and evaluation: Pharmaceutical assays’, Springer-VerlagBerlin, 1997). For example, drug screening in animal models, in vitrotests using animal cells, or in vivo tests involving toxicology tests inanimals. An in vitro model can be used for screening libraries ofcompounds in any of a variety of drug screening techniques.

Candidate agents encompass numerous chemical classes, though typicallythey are organic molecules, preferably small organic compounds having amolecular weight of more than 50 and less than about 2,500 Daltons.Candidate agents comprise functional groups necessary for structuralinteraction with proteins, particularly hydrogen bonding, and typicallyinclude at least an amine, carbonyl, hydroxyl or carboxyl group,preferably at least two of the functional chemical groups. The candidateagents often comprise carbocyclic or heterocyclic structures and/oraromatic or polyaromatic structures substituted with one or more of theabove functional groups.

Candidate agents are also found among biomolecules including peptides,amino acids, saccharides, fatty acids, steroids, purines, pyrimidines,nucleic acids and derivatives, structural analogs or combinationsthereof. Candidate agents are obtained from a wide variety of sourcesincluding libraries of synthetic or natural compounds. For example,numerous means are available for random and directed synthesis of a widevariety of organic compounds and biomolecules, including expression ofrandomized oligonucleotides and oligopeptides. Alternatively, librariesof natural compounds in the form of bacterial, fungal, plant and animalextracts are available or readily produced. Additionally, natural orsynthetically produced libraries and compounds are readily modifiedthrough conventional chemical, physical and biochemical means, and maybe used to produce combinatorial libraries. Known pharmacological agentsmay be subjected to directed or random chemical modifications, such asacylation, alkylation, esterification, amidification, etc. to producestructural analogs.

Another technique for drug screening, which may be used, provides forhigh throughput screening of compounds having suitable binding affinityto the protein of interest as described in published PCT application WO84/03564. In this method, as applied to the proteins of the inventionlarge numbers of different small test compounds, e.g. aptamers,peptides, low-molecular weight compounds etc., are provided orsynthesized on a solid substrate, such as plastic pins or some othersurface. The test compounds are reacted with the proteins or fragmentsthereof, and washed. Bound proteins are then detected by methods wellknown in the art. Purified proteins can also be coated directly ontoplates for use in the aforementioned drug screening techniques.Alternatively, non-neutralizing antibodies can be used to capture thepeptide and immobilize it on a solid support. In another embodiment, onemay use competitive drug screening assays in which neutralizingantibodies capable of binding the protein specifically compete with atest compound for binding the protein. In this manner, the antibodiescan be used to detect the presence of any peptide, which shares one ormore antigenic determinants with the protein.

The present invention further particularly provides a method, whereinthe pharmaceutical composition to be produced further comprisesneuroprotective substances, nootrophic substances, brilliant blue,piperidine or derivatives thereof, adamantine derivatives, substitutedphenyl compounds, oxidized ATP,2-O-(4-benzoylbenzoyl)adenosine-5-triphosphate or3-O-(4-benzoylbenzoyl)adenosine-5-triphosphate. It is also envisagedthat the pharmaceutical compositions to be produced further compriseantidepressants such as fluoxetine, paroxetine, sertraline, fluroxamine,mirtazapine, reoretine, nefazodone or lithium carbonate.

In a preferred embodiment of the present invention, the compounds of theaforementioned methods comprise antagonist(s), partial antagonist(s),partial agonist(s) and/or agonist(s) for an altered ATP-gated ionchannel P2X7R.

In accordance with the present invention, the term “antagonist” denotesmolecules/substances, which are capable of inhibiting and/or reducing anagonistic effect. The term “antagonist” comprises competitive,non-competitive, functional and chemical antagonists as described, interalia, in Mutschler, “Arzneimittelwirkungen” (1986), WissenschaftlicheVeriagsgesellschaft mbH, Stuttgart, Germany. The term “partialantagonist” in accordance with the present invention means amolecule/substance that is capable of incompletely blocking the actionof agonists through, inter alia, a non-competitive mechanism.

In the context of the present invention, an antagonist is preferably adrug that does not provoke a response itself, but blocksagonist-mediated responses. It is a chemical entity that opposes thereceptor-associated responses normally induced by another bioactiveagent. For the P2X7R, the antagonists have an IC50 between 10 nanomolarand 300 micromolar.

As “agonist”, in accordance with this invention, molecules/substancesare denoted which have an affinity as well as an intrinsic activity.Mostly, said intrinsic activity (α) is defined as being proportional tothe quotient of the effect, triggered by said agonist (EA) and theeffect which can be maximally obtained in a given biological system(Emax): therefore, the intrinsic activity can be defined as$\left. \alpha \right.\sim\frac{E_{A}}{E_{\max}}$

The highest relative intrinsic activity results from EA/Emax=1. Agonistswith an intrinsic activity of 1 are full agonists, whereassubstances/molecules with an intrinsic activity of >0 and <1 are partialagonists. Partial agonists show a dualistic effect, i.e. they compriseagonistic as well as antagonistic effects.

Preferably, in the context of the present invention, an agonist (or fullagonist) is an endogenous substance or a drug that can interact with areceptor and initiate a maximal or complete physiological or apharmacological response characteristic of that receptor. ATP, thenatural ligand for the P2X7R, is an agonist with an EC50 of 300micromolar while the synthetic P2X7R agonist Bz-ATP has an EC50 of 8micromolar. Thus, agonists of P2X7R have an EC50 equal or below 300micromolar. The EC50 is defined as the concentration of agonist thatprovokes a response half way between the baseline response and maximumresponse on a dose response curve where the X-axis plots concentrationof an agonist and the Y-axis plots ion current. An inverse agonist (alsocalled negative antagonist) is a drug which acts at the same receptor asthat of an agonist, yet produces an opposite effect. A partial agonistis an endogenous substance or a drug that also provokes physiological ora pharmacological response but, the maximum response is less than themaximum response to a full agonist, regardless of the amount of drugapplied. In the case of P2X7R, partial agonists have EC50s higher than300 micromolar.

The person skilled in the art can, therefore, easily employ thecompounds and the methods of this invention in order to elucidate theagonistic and/or antagonistic effects and/or characteristics of acompound/molecule/substance to be identified and/or characterized inaccordance with any of the above described methods. Preferably, anidentified antagonist of the ATP-gated ion channel P2X7R comprising themutation(s) and/or deletion(s) described hereinabove may be useful toreestablish the properties normally shown by wild-type P2X7R ATP-gatedion channels. An identified agonist of the ATP-gated ion channel P2X7Rcomprising the mutation(s) and/or deletion(s) described hereinabove maybe useful to reestablish the lost functionality of the P2X7R ATP-gatedion channel.

The Figures show:

FIG. 1 a. Genomic map of the region on the human chromosome 12associated to bipolar affective disorder. Genes found between markersNBG11 and NBG2 are depicted.

FIG. 1 b. Graphic illustrating the multipoint analysis using ASPEX onindependent sib-pairs.

FIG. 1 c. Graphic illustrating the multipoint analysis using ASPEX onall sib-pairs

FIG. 1 d. Graphic illustrating the ASPEX sib_Phase by considering onlyindependent sib-pairs

FIG. 1 e. Graphic illustrating the ASPEX sib_phase by considering allsib-pairs

FIG. 1 f. Effect of the P2XR7v13A polymorphism on basal cortisol levelsbefore and after administration of dexamethasone (DST test). Individualswere subjected to the test within the first ten days of admission.Individuals with the AG and GG genotypes have significantly lowercortisol levels pre- and post-dexamethasone administration.

FIG. 1 g. Effect of the P2XR7v13A polymorphism on cortisol responseduring the Dex/CRH test. Individuals were subjected to the test withinthe first ten days of admission (i.e. At admission) and at the last tendays before discharge (i.e. at discharge). Individuals with the GGgenotype have lower cortisol levels in response to the Dex/CRH test atadmission and at discharge. These results are indicative of an abnormalHPA axis.

FIG. 1 h. Effect of the P2XR7v13A polymorphism on ACTH response duringthe Dex/CRH test. Individuals were subjected to the test within thefirst ten days of admission (i.e. at admission) and at the last ten daysbefore discharge (i.e. at discharge). Individuals with the GG genotypehave lower ACTH levels in response to the Dex/CRH test, at admission andat discharge. These results are indicative of an abnormal HPA axis.

FIG. 1 i. Duration of antidepressant treatment until remission.Depression is diagnosed according to the Hamilton Depression RatingScale (HAM-D; Hamilton, Br. J. Soc. Clin. Psychol. 6 (1967) 278-296). AHAM-D score of 10 or below is regarded as remission of the depressivesymptoms.

FIG. 1 j. Effect of the P2XR7v13C polymorphism on basal cortisol levelsbefore and after administration of dexamethasone (DST test). Individualswere subjected to the test within the first ten days of admission.Individuals with the CC genotypes have elevated cortisol levelspost-dexamethasone administration.

FIG. 1 k. Effect of the P2XR7v13C polymorphism on cortisol responseduring the Dex/CRH test. Individuals were subjected to the test withinthe first ten days of admission (i.e. at admission) and at the last tendays before discharge (i.e. at discharge). Individuals with the AC or CCgenotype have elevated cortisol levels in response to the Dex/CRH testat admission, indicating an abnormal HPA axis.

FIG. 1 l. Effect of the P2XR7v13C polymorphism on ACTH response duringthe Dex/CRH test. Individuals were subjected to the test within thefirst ten days of admission (i.e. at admission) and at the last ten daysbefore discharge (i.e. at discharge). Individuals with the CC genotypehave lower ACTH levels in response to the Dex/CRH test, at admission andat discharge. These results are indicative of an abnormal HPA axis.

FIG. 2. RT-PCR analysis of the complete coding sequence of P2X7R indifferent tissues

FIG. 3. P2X7R expression in the olfactory bulb, hypothalamus andependymal cells in the brain of a stress-free mouse. Magnification 100×.

FIG. 4. P2X7R expression in the hippocampus/dentate gyrus andsubcommisural organ in the brain of a stress-free mouse. Magnification100×.

FIG. 5. Floating behaviour in the forced swim test. Passive stresscoping behaviour decreased after long-term treatment with theantidepressant paroxetine (Par28: treated with paroxetine for 28 days,per os). Basal n=8; vehicle n=8; Par28 n=8.

FIG. 6. Comparative analysis of P2X7R expression in the olfactory bulbof stress-free, vehicle-treated and antidepressant-treated mice.Magnification 100×.

FIG. 7. Comparative analysis of P2X7R expression in the hypothalamus ofstress-free, treated-treated and antidepressant-treated mice.Magnification 100×.

FIG. 8. Comparative analysis of P2X7R expression in ependymal cells ofstress-free, vehicle-treated and antidepressant-treated mice.Magnification 100×.

FIG. 9. Comparative analysis of P2X7R expression in the hippocampus ofstress-free, vehicle-treated and antidepressant-treated mice.Magnification 25×.

FIG. 10. P2X7R expression in the hippocampus of a vehicle treated mouse.Magnification 25×.

FIG. 11. P2X7R expression in the hippocampus of a mouse treated with theantidepressant paroxetine. Magnification 25×.

FIG. 12. Detailed expression of P2X7R in the dentate gyrus of a mousetreated with the antidepressant paroxetine. Magnification 400×.

FIG. 13. Comparative analysis of P2X7R expression and apoptotic cells inthe hippocampus of a mouse treated with the antidepressant paroxetine.Magnification 100×.

FIG. 14. Floating behaviour in the forced swim test. Passive stresscoping behaviour increased after acute intrahippocampal (bilateral,dentate gyrus) of siRNA targeting P2X7R. Vehicle n=7; control RNA n=10;P2X7R siRNA n=9.

FIG. 15. Comparative analysis of P2X7R expression in the hippocampus ofmice treated with vehicle, control RNA and of siRNA targeting P2X7R.Magnification 100× upper row, 25× lower row.

FIG. 16 a, b, c, d, e. Three splicing variants caused by polymorphismsin the introns of P2X7R.

FIG. 17. Expression of P2X7R in immortalized hippocampal cell lines.

FIG. 18. Increase calcium influx in hippocampal cells treated with aP2X7R agonist compound (BzATP).

FIG. 19 a, b. Entry of ethidium bromide dye into hippocampal cells (a)treated with P2X7R agonist compound (BzATP) or (b) pre-treated with aP2X7R antagonist compound.

FIG. 19 c. Agonist action of BzATP and tenidap on P2X7R activity. Thecalcium channel activity of human P2X7R was measured under basalconditions for four seconds to 10 seconds. A. Negative controlconsisting of cells loaded with 10 μM Fluo-4-AM without furthertreatment. B. Cells treated with 20 μM BzATP after four seconds of basalmeasurement. C. Cells treated with 50 μM tenidap after four seconds ofbasal measurement.

FIG. 20. Effect of intrahippocampal injection of a P2X7R agonistcompound (BzATP) on behaviour in the forced swim test.

FIG. 21. Open field test measuring locomotor activity of mice treatedwith a P2X7R agonist compound (BzATP).

FIG. 22. Comparative analysis of apoptotic cells in the hippocampus of amouse treated with control vehicle solution or a P2X7R agonist compound(BzATP).

FIG. 23. Effect of intrahippocampal injection of the P2X7R antagonistKN-62 and oATP on behaviour during the forced swim test

FIG. 24. Open field test measuring locomotor activity of mice treatedwith the P2X7R antagonist KN-62 and oATP.

A better understanding of the present invention and of its manyadvantages will be had from the following examples, offered forillustrative purposes only, and are not intended to limit the scope ofthe present invention in any way.

EXAMPLE 1 Linkage Analysis of Bipolar Affective Disorder in aHomogeneous Human Population

41 families of different sizes containing a total of 485 sampledindividuals from the region of Saguenay/Lac St-Jean were used in thelinkage analysis. Individuals were distributed according to theirdiagnoses as follows: 105 individuals afflicted with Bipolar Disordertype I (BPI) or schizoaffective disorder bipolar type; 42 individualsdiagnosed with Bipolar Disorder type II (BPII); 54 individuals withrecurrent major depression; and 57 individuals with single episode majordepression. The remaining 227 individual were unaffected and normal. Forthe purpose of the calculation, the following classification was used:individuals diagnosed with either BPI, schizoaffective disorder, bipolartype, BPII and recurrent major depression were considered as affected(n=201); individuals with a single major depression episode were scoredas unknown phenotype (n=57); and all others diagnoses as unaffected(n=227).

Blood samples from each individual were collected in 10-ml K3 EDTAVacutainer tube (Becton-Dickinson) and genomic DNA was isolated byPuregene DNA Isolation kit (Gentra Systems). Blood was poured into 50 mlconical tube and diluted with four volume of Red Blood Cell LysisSolution. After an incubation of 10 minutes at room temperature, thetube was centrifuged for 10 minutes at 2,000 g and supernatant wasremoved leaving behind cell pellet and 200-400 μl of the residualliquid. Cells were resuspended by vortexing the tube and 9 ml of CellLysis Solution were added with up and down pipetting. 40 μl of RNAse ASolution (20 mg/ml) were added and the sample was mixed by inverting thetube several times. Sample was incubated at 37° C. for 15 minutes andcooled to room temperature. 3 ml of Protein Precipitation Solution wereadded to cell lysate. Tube was vigorously vortexed for 30 seconds andcentrifuged at 2,000 g for 10 minutes. Supernatant was poured into a newtube containing 9 ml of 100% isopropanol. Sample was mixed by invertinggently several times. Tube was centrifuged at 2,000 g for 5 minutes. TheDNA white pellet was washed with 10 ml 70% ethanol and the tube wascentrifuged at 2,000 g for 3 minutes. Ethanol was poured off and pelletallowed to partially air dry. DNA was solubilized in 500 μl of DNAHydration Solution. Final concentration was adjusted to 300-400 μg/ml.

A fluorescent-based method was used for the genotyping of microsatellitemarkers. Briefly, the region encompassing each repeated sequence wasamplified by PCR using an unlabeled primer and a fluorescent-labeledprimer (Applied biosystems inc, CA, USA). The marker-associated dyes andthe corresponding PCR product length are listed in table 2. The PCRreaction was performed using 10 ng of DNA sample, 0.2 unit of Taqplatinum DNA polymerase (Invitrogene, CA, USA), 20 mM Tris-Cl (pH 8.4),50 mM KCl, 1.5 mM MgCl₂, 100 μM of dNTP, and 1.5 μM of each primer in afinal volume of 7 μl. The samples were incubated at 95° C. for 3 minutesto activate the Taq platinum DNA polymerase, then 10 cycles of PCRamplification were performed as follows: 95° C. for 15 seconds; 58° C.for 15 seconds; 72° C. for 30 seconds; after that 15 cycles wereperformed as follow: 89° C. for 15 seconds; 58° C. for 15 seconds; 72°C. for 30 seconds. Finally, the samples were incubated at 72° C. for 30min. Following the PCR amplification samples were pooled according totheir dye-labeled primer and their PCR product length (pool of foursamples). Pooled sample were separated on an ABI 3100 DNA analyzer(Applied Biosystems inc, CA, USA). The resulting data were analysedusing Genemapper2 (Applied Biosystems inc, CA, USA), and compiled in a4D database (ACIUS) designed in a Macintosh environment as previouslydescribed (Morissette et al., Am. J. Med. Genet. (Neuropsychiatr.Genet.) 88 (1999), 567-587)

Markers used in the following linkage analysis are shown in table 2.Recombination fraction (q) between successive markers was computedaccording to the analyzed families. TABLE 2 Genomic markers used for thelinkage analysis Associated Allele length Distance Cumulative distanceHeterozygosity Locus dye (bp) (q) (cM) (%) D12S1619 VIC 170-210 0.01350.00 74.5 NBG11 VIC 204-218 0.006 1.37 65.5 D12S1666 FAM 241-281 0.0011.97 66.9 NBG5 VIC 253-261 0.001 2.07 38.3 D12S1721 VIC 263-299 0.0052.17 72.1 NBG8 VIC 166-188 0.011 2.67 73.3 NBG6 NED 182-218 0.0115 3.7973.9 NBG9 VIC 156-180 0.0035 4.95 68.9 NBG10 FAM 174-186 0.001 5.30 49.7NBG12 NED 165-207 0.009 5.40 64.2 NBG4 NED 171-199 0.001 6.31 66.4 NBG3VIC 182-206 0.006 6.41 64.8 NBG2 VIC 171-199 7.01 54.2

-Haldane's map function was used for cumulative distance in cMorgans.

For bipoint parametric analysis, MOD score analysis were used whereparametric LOD score were maximized over genetic models.

The following results were obtained under MOD score analysis forrecessive models. TABLE 3 MOD score analysis for recessive modelsDistance Cumulative distance Locus (q) (cM) LOD score (q_(max)) D12S16190.0135 0.00 3.46 (0.10) NBG11 0.006 1.37 4.06 (0.04) D12S1666 0.001 1.971.22 (0.14) NBG5 0.001 2.07 0.66 (0.16) D12S1721 0.005 2.17 2.82 (0.10)NBG8 0.011 2.67 1.51 (0.00) NBG6 0.0115 3.79 4.77 (0.06) NBG9 0.00354.95 0.75 (0.22) NBG10 0.001 5.30 0.74 (0.00) NBG12 0.009 5.40 1.41(0.16) NBG4 0.001 6.31 3.56 (0.08) NBG3 0.006 6.41 3.96 (0.08) NBG2 7.012.59 (0.10)

Model-free LOD score studies using ANALYZE, sib_phase from the ASPEXV1.85 package (David Hinds and Neil Risch 1999;ftp://lahmed.stanford.edu/pub/aspex, see alsohttp://watson.hgen.pitt.edu/docs/_usage.html) and SIMWALK2 (Sobel andLange, Am J Hum Genet 58 (1996), 1323-1337) were performed to analyzethe allele sharing among affected sib-pairs. The ANALYZE program weightssibships according to their size. The ASPEX sib_phase program usesallele frequencies to reconstruct missing information, and is tailoredfor data sets where parents are missing, but additional typed childrenmay be used to reconstruct and phase the parents. SimWalk2 is astatistical genetics computer application for haplotype, parametriclinkage, non-parametric linkage (NPL), identity by descent (IBD) andmistyping analyses on any size of pedigree. SimWalk2 uses Markov chainMonte Carlo (MCMC) and simulated annealing algorithms to perform thesemultipoint analyses.

ASPEX sib_phase was used with two computational strategies: First, byusing strictly independent sib pairs; secondly, by using all affectedsib-pair combinations. ASPEX was performed for bi-point and multipointcalculations.

The bi-point results observed with ANALYZE and ASPEX are shown in Table4. TABLE 4 Bi-point results observed with ANALYZE and ASPEX CumulativeSib-pair from sib_phase sib_phase distance ANALYZE LOD score LOD scoreLocus Distance (q) (cM) LOD score indep. sib-pairs all sib-pairsD12S1619 0.0135 0.00 2.31 2.55 3.14 NBG11 0.006 1.37 2.83 2.72 3.27D12S1666 0.001 1.97 1.01 2.52 3.14 NBG5 0.001 2.07 0.50 2.52 3.13D12S1721 0.005 2.17 1.57 2.51 3.12 NBG8 0.011 2.67 0.51 2.24 2.75 NBG60.0115 3.79 2.55 2.11 2.64 NBG9 0.0035 4.95 0.49 1.65 1.97 NBG10 0.0015.30 0.77 1.45 2.10 NBG12 0.009 5.40 0.47 1.44 2.17 NBG4 0.001 6.31 1.211.29 3.07 NBG3 0.006 6.41 1.84 1.29 3.07 NBG2 7.01 1.24 1.22 3.00

SIMWALK2 computed four different statistics based on descent trees.These statistics measure the degree of clustering among the markeralleles descending from the founders.

Statistic A is the number of different founder-alleles contributingalleles to the affected it is most powerful at detecting linkage to arecessive trait. Statistic B is the maximum number of alleles among theaffected descended from any one founder-allele and most powerful atdetecting linkage to a dominant trait. Statistic C is the ‘entropy’ ofthe marker alleles among the affected. Statistic D is the extent ofallele sharing among all affected pairs as measured by their IBD kinshipcoefficient. Statistics C and D are more general statistics indicatingwhether a few founder-alleles are overly represented among the affected.

Table 5 shows the results observed with SIMWALK2. The authors signalthat p-values should be generally conservative. They are expressed as−Log(p-values). For correspondence purpose, −Log(0.05)=1.30,−Log(0.01)=2, −Log(0.001)=3 etc. TABLE 5 SIMWALK2 analysis Cumul.Distance Distance STAT(A) STAT(B) STAT(C) STAT(D) Locus (q) (cM)−Log(p-value) −Log(p-value) −Log(p-value) −Log(p-value) D12S1619 0.01350.00 1.4550 0.4103 1.1306 1.1310 NBG11 0.006 1.37 2.0157 1.4375 1.59551.9845 D12S1666 0.001 1.97 2.0236 0.9765 1.4727 1.4614 NBG5 0.001 2.071.7596 0.8558 1.3866 1.3602 D12S1721 0.005 2.17 1.6628 1.1692 1.42351.6384 NBG8 0.011 2.67 1.5374 0.6940 1.0623 1.1552 NBG6 0.0115 3.791.5896 0.4452 1.0935 1.1786 NBG9 0.0035 4.95 1.2677 0.3815 0.8412 0.9133NBG10 0.001 5.30 1.1117 0.3642 0.6987 0.7554 NBG12 0.009 5.40 1.08090.3485 0.6694 0.7179 NBG4 0.001 6.31 1.1024 0.4148 0.6368 0.8544 NBG30.006 6.41 1.1040 0.4146 0.6373 0.8559 NBG2 7.01 1.0963 0.5380 0.65870.9356

Multipoint result observed with ASPEX when only independent sib-pairswere used (FIG. 1 b). The maximum LOD score value was observed at NBG11.

Multipoint result observed with ASPEX when all sib-pairs were considered(FIG. 1 c). The maximum LOD score value was observed at NBG11 but asecond peak appeared at NBG4 and NBG3.

Multipoint and bi-point LOD score values computed by ASPEX were similar.The second peak, observed when all sib-pairs are used, may be explainedby the presence of a recombinant affected individual, with many affectedsibs, sharing the chromosomal region telomeric to NBG12. This kind ofindividuals has a large impact on LOD score values when all sib-pairsare used instead of one sib-pair. This situation was observed in twosibships.

Strata analysis was subsequently performed. Although HOMOG did notdetect evidence for heterogeneity, a homogeneity test was constructedbased on allele sharing found in selected chromosomal regions. Only 20of the 41 families were used for this analysis since the others were notgenotyped in all these regions. For each marker within the selectedregions, the proportion of alleles shared IBD by affected sib-pairs wasestimated with ASPEX (sib_phase). For each region retained, theproportion of shared alleles was used as variable for a PrincipalComponent Analysis and the first principal component as an index oflinkage. Correlation analysis was done on these indexes to detectheterogeneity (correlation <0) or epistasis (correlation >0). Fisheralgorithm was used to classify into two groups of families as linked orunlinked to a particular locus. A negative correlation was observedbetween the chromosome 12 region and the chromosome 15 area (r=−0.51;p=0.023). Cluster analysis suggested that 11 families out of 20 werelinked to chromosome 12. This sub-sample was called the strata.

This strata included 11 families (266 sampled individuals) that include52 BPI or schizoaffective disorder, bipolar type, 20 BPII and 28recurrent major depression

The following MOD score values illustrated in Table 6 were obtainedunder recessive models. TABLE 6 MOD scores under recessive modelsDistance Cumulative distance Locus (q) (cM) LOD score (q_(max)) D12S16190.0135 0.00 4.03 (0.08) NBG11 0.006 1.37 4.98 (0.00) D12S1666 0.001 1.971.49 (0.12) NBG5 0.001 2.07 0.79 (0.14) D12S1721 0.005 2.17 4.23 (0.06)NBG8 0.011 2.67 2.79 (0.00) NBG6 0.0115 3.79 5.06 (0.06) NBG9 0.00354.95 1.57 (0.14) NBG10 0.001 5.30 1.73 (0.00) NBG12 0.009 5.40 1.65(0.12) NBG4 0.001 6.31 4.60 (0.08) NBG3 0.006 6.41 4.84 (0.06) NBG2 7.012.80 (0.06)

Model-free LOD score results obtained with ANALYZE and ASPEX applied tothe strata are shown in Table 7. TABLE 7 Model-free LOD score obtainedwith ANALYZE and ASPEX sib_phase Cumulative LOD score sib_phase distanceANALYZE independent sib- LOD score Locus Distance (q) (cM) LOD scorepairs all sib-pairs D12S1619 0.0135 0.00 4.54 5.29 7.65 NBG11 0.006 1.374.29 5.34 7.70 D12S1666 0.001 1.97 2.77 5.36 7.74 NBG5 0.001 2.07 0.675.36 7.74 D12S1721 0.005 2.17 4.48 5.35 7.74 NBG8 0.011 2.67 2.97 4.877.00 NBG6 0.0115 3.79 4.05 4.59 6.76 NBG9 0.0035 4.95 2.03 3.72 5.41NBG10 0.001 5.30 2.00 3.42 5.89 NBG12 0.009 5.40 0.89 3.44 6.11 NBG40.001 6.31 2.84 3.71 9.00 NBG3 0.006 6.41 3.89 3.71 9.01 NBG2 7.01 1.913.52 8.73

Model-free results observed with SIMWALK2 are illustrated in Table 8.TABLE 8 Model-free LOD score obtained with SIMWALK2 Cumulative DistanceDistance STAT(A) STAT(B) STAT(C) STAT(D) Locus (q) (cM) −Log(p-value)−Log(p-value) −Log(p-value) −Log(p-value) D12S1619 0.0135 0.00 2.59630.9565 3.2156 2.3584 NBG11 0.006 1.37 3.0698 1.7400 3.7747 3.0103D12S1666 0.001 1.97 2.9340 1.6546 3.5812 2.7223 NBG5 0.001 2.07 2.97811.2722 3.6505 2.7846 D12S1721 0.005 2.17 2.9680 1.2630 3.6844 2.7752NBG8 0.011 2.67 3.0954 1.0804 3.4399 2.5654 NBG6 0.0115 3.79 3.16321.0672 3.2670 2.5956 NBG9 0.0035 4.95 2.2106 1.0137 2.7765 2.4456 NBG100.001 5.30 2.5513 1.0251 2.7625 2.1914 NBG12 0.009 5.40 2.4893 0.98682.6841 2.0920 NBG4 0.001 6.31 2.9028 1.1312 3.4063 2.8156 NBG3 0.0066.41 2.9070 1.1326 3.4637 2.8300 NBG2 7.01 2.8430 1.1108 3.3135 2.7978

Multipoint results on the strata with ASPEX sib_phase by consideringonly independent sib-pairs (FIG. 1 b) or all sib-pairs (FIG. 1 c) areshown in FIGS. 1 d and 1 e. As previously reported a second peakappeared when all sib-pairs were observed.

A confidence interval was calculated. GENEFINDER (Liang et al., Am. J.Hum. Genet. 66 (2000), 1631-1641) was used to estimate the location ofthe susceptibility gene (say t). The method is based on the IBD(Identity by Descent) sharing of affected sib-pairs for multiplemarkers. For the purpose of our analysis, pedigrees were divided intosibship. 56 nuclear families and 183 sib-pairs were used. Liang K Y,Huang C Y, Beaty T H (2000) A unified sampling approach for multipointanalysis of qualitative and quantitative traits in sib pairs. Am J HumGenet 66:1631-1641

The GENEFINDER results points to localization of a susceptibility genefor affective disorders at 3.19±0.446 cM telomeric to the markerD12S1721 (D12S1721 is approximately located at 136.82 cM on thesex-averaged Marshfield chromosome 12 map).

95% C.I.: [2.32, 4.06];

99% C. I.: [2.03, 4.35]

99.9% C. I.: [1.71, 4.67]

From the strata, 24 nuclei, and 107 sib-pairs were obtained, and thelocation of the susceptibility gene was estimated at 3.07±0.57 (see mapabove). The following confidence interval (C.I.) was obtained:

95% C.I.: [1.95, 4.19];

99% C. I.: [1.59, 4.55]

99.9% C. I.: [1.18, 4.96]

An association study using the NBG microsatellite markers was done withCLUMP (Sham & Curtis, Ann. Hum. Genet. 59 (1995), 97-105). Samples weredistributed as follow: 83 male/case; 124 female/case; 95 male/control;and 101 female/control. One thousand simulations were used to estimatep-values. The observed results are summarized in Table 9. TABLE 9Association study using the NBG microsatellite Sample T1statisticT2statistic T3statistic (p- Locus Case Control (p-value) (p-value)value) T4statistic (p-value) NBG11 204 129 0.226 0.562 0.410 0.421 NBG5206 194 0.972 0.980 0.948 0.971 NBG8 206 194 0.983 1.000 0.994 0.978NBG6 206 194 0.147 0.074 0.759 0.485 NBG9 206 190 0.512 0.940 0.7860.583 NBG10 206 190 0.594 0.480 0.403 0.709 NBG12 206 190 0.002 0.0190.003 0.117T1 statistic is the usual chi-squared statistic on the raw contingencytableT2 statistic is the usual chi-squared statistic apply on he contingencytable obtained after collapsing columns with small expected valuestogetherT3 statistic is the largest chi-squared statistic got by comparing onecolumn of the original table against the total of the others columnsT4 statistic is the largest chi-squared statistic got by comparing anycombination of alleles against the rest.

Only the NBG12 marker gave significant association at the 1% level. Forthe others markers, there was no single alleles that seems to beassociated with bipolar disorder. It seems that no founder-alleles wasoverly represented among the affected. There is no significant resultfor association of genotypes with the NBG markers.

Further microsatellite marker based association studies using CLUMP wasperformed on samples containing additional control and case individuals.One thousand simulations were used to estimate p-values. TABLE 9aEmpirical p-values observed with CLUMP for statistics T1 and T3 forallelic and genotypic analyses of microsatellite markers AllelesGenotypes T1 T3 T3 Effective (p- (p- T1 (p- Name case controls value)value) (p-value) value) NBG11 204 98 0.250 0.421 0.680 0.553 D12S1666208 175 0.366 0.543 0.393 0.476 NBG5 213 179 0.969 0.934 0.997 1.000D12S1721 210 176 0.693 0.463 0.805 0.838 NBG8 213 179 0.754 0.921 0.9730.929 NBG6 213 179 0.008 0.356 0.172 0.449 NBG9 213 175 0.759 0.7680.690 0.606 NBG10 213 175 0.521 0.178 0.122 0.173 D12S1349 212 180 0.8870.864 0.782 0.816 NBG12 213 175 0.002 <10⁻³ 0.018 0.552 NBG4 207 1780.418 0.506 0.813 0.545 NBG3 209 175 0.171 0.829 0.601 0.897 D12S378 211180 0.171 0.405 0.540 0.560 NBG2 210 170 0.896 0.749 0.210 0.613D12S1614 210 179 0.803 0.692 0.710 0.831 D12S342 211 180 0.394 0.7400.445 0.622 D12S340 209 179 0.890 0.869 0.895 0.838 D12S1639 209 1800.087 0.170 0.652 0.295 D12S1634 211 181 0.361 0.248 0.505 0.590D12S2075 203 181 0.023 0.157 0.085 0.451

HWE hypothesis was satisfied at the 5% level for each microsatellitemarker after application of the conservative Bonferroni corrections formultiple testing (Bland & Altman, Brit. J. Med. 310 (1995) 170). Table9a lists empirical p-values observed with CLUMP for allele and genotypeassociation analyses. Empirical p-values less than 0.005 were observedat marker NBG12 for T1 and T3 statistics under allelic associationanalysis. T1 statistic suggested allelic association between bipolaraffective disorders and NBG6 (empirical p-value=0.008). Moreover, abarely significant empirical p-value of 0.023 was observed at the mostdistal marker D12S2075.

In conclusion, the parametric and model-free multipoint results suggestto investigate genes located between D12S1619 and D12S1666. Moreover,according to GENEFINDER results, genes situated centromeric to NBG9should be considered for association and linkage disequilibriumanalysis. Moreover, positive association was seen with the NBG6 marker,which is located in intron 9 of the P2X7R gene.

EXAMPLE 2 Physical Mapping and Mutation Analysis of Chromosome 12Associating the P2X7R to Bipolar Affective Disorders

The most conservative prediction for the disease-associated region isincluded between markers NBG11 and NBG2 (see FIG. 1 a). This region wasdelimited according to linkage and association analysis described inExample 1, using genethon markers and NBG markers. The approximatelength of this region is 5.2 Mb. Two major gaps (between FLJ10701 andFLJ32372, and between FLJ1466 and MONDOA) were included in this region.At least 73 genes were listed in this area, where 48 are known genes and25 are unknown but associated to mRNA and/or EST clusters based on thelast genome assembly available at UCSC (November 2002). Predicted geneswere not listed. However, the estimation of Cl 99% (confidence interval)using GENEFINDER has limited the most interesting region between markersD12S1666 and NBG9. This genomic region covers 1.6 Mb and includes atleast 28 genes, and has no major gap. Thus, the term fBAD (familialBipolar Affective Disorders) region was used to describe the genomicsegment between D12S1666 and NBG9. Genes found within this regioninclude CaMKK2, CABP, P2X7, P2X4, PIN, PLA2, G1B, CIT, PXN, Rab35, andAPC5. However, given the present art, it would not have been obvious toan ordinary person skilled in the art to select P2X7R as the geneassociated with affective diseases. Other genes from the ones listedabove would be obvious.

For example, the CaMKK2 gene (also known as Ca²⁺/Calmodulin-dependentprotein kinase kinase beta, or CaMKKb) is a serine/threonine proteinkinase involved in Ca²⁺ dependent signalling pathways. CaMKK2 canactivate in vitro the downstream kinases CaMKIV and CaMKI, whichmodulate gene transcription through phosphorylation of transcriptionfactors (e.g., CREB, SRF, MEF2; Corcoran and Means, J. Biol. Chem. 276(2001), 2975-2978; Soderling, Trends Biochem. Sci. 24 (1999), 232-236).Its role in the Ca²⁺ cascade is not critical. Some studies suggest thatCaMKs could be activated without the CaMKKs phosphorylation (Matsushitaand Nairn, J. Biol. Chem. 274 (1998), 10086-10093). However, CaMKKphosphorylation step would contribute to amplification of the Ca²⁺signal since CaMKK is more sensitive to activation by Ca²⁺/Calmodulin,therefore CaMKK would be an important mediator when the levels ofintracellular Ca²⁺ are low (Anderson et al., J. Biol. Chem. 273 (1998),31880-31889).

CaMKK2 is an obvious target for depression since prior art suggest thatcAMP-dependent signaling pathways (mediated by PKA activation) isaffected in brain from patients with Bipolar Affective Disorders (Fieldet al., J. Neurochem. 73 (1997), 1704-1710; Rahman et al., J. Neurochem.68 (1997), 297-304; Takahashi et al., J. Neurosci. 19 (1999), 610-618).According to a study using lymphoblastic cell lines, Bipolar disordercould be related to a elevated intracellular calcium levels (Yoon etal., Mol. Psychiatry 6 (2001), 678-683). Moreover, some groups foundrelations between antidepressant drugs and CaMK activation (Budziszewskaet al., Br. J. Pharmacol. 130 (2000), 1385-1393; Consogno et al.,Neuropsychopharmacology 24 (2001), 21-30; Mori et al., Neuropharmacology40 (2001), 448-456; Zanotti et al., Neuropharmacology 37 (1998),1081-1089). Furthermore, inhibition of CaMKK by PKA-mediatedphosphorylation suggest a close relationship between both pathways(Matsushita et al., J. Biol. Chem. 273 (1999), 21473-21481). Theseobservations would suggest to a person skilled in the art that CaMKK2 isthe gene responsible for bipolar affective disease.

Another obvious candidate for affective disorders would have been theCABP1 gene which generates four neuronal Ca²⁺-binding protein byalternative usage of the 9 coding exons, which are L-CABP, S-CABP,calbrain, and caldendrin (Haeseleer et al., J. Biol. Chem. 275 (2000),1247-1260). Their expression is almost totally restricted to braintissues. A functional study on calbrain reveals its negative effect onCa²⁺/Calmodulin-dependent CaMKII activity by competitively interactswith the CaM-binding domain of CaMKII (Yamagushi et al., J. Biol. Chem.274 (1999), 3610-3616). One would expect similar roles in Ca²⁺ signalingfor other CABP1 alternative products. Participation of CABP1 gene inCa²⁺-dependent signaling pathways would make it obvious to one skilledin the art to select this gene as a candidate for bipolar affectivedisorder. However, all CABP1 exons were analyzed for the presence ofmutations, and surprisingly only two mutations were detected innoncoding regions.

The PIN gene (Protein inhibitor of NOS (Nitric oxide synthase)) isanother obvious candidate responsible for bipolar affective disorder.Nitric oxide (NO) in the brain, may be involved in apoptosis,synaptogenesis, and neuronal development. Because NO cannot be stored invesicles like other neurotransmitters, its release is regulated by theactivity of NOS (Nitric oxide synthase). PIN is a direct inhibitor ofNOS by binding and destabilizing the active homodimer complex of NOS(Jaffrey et al., Science 274 (1996), 774-777). PIN is highly conservedthroughout the evolution and is expressed in many cell types. A recentclinical study evaluating plasma nitrate levels in depressive statessuggests that NO production is increased in depression (Suzuki et al.,J. Affect. Disord. 63 (2001), 221-224) and may result from a deficiencyin NOS inhibition. Moreover in a mouse model, NO synthase antagonistshave been linked to antidepressant properties (Harkin et al., 1999;Karolewicz et al., Eur. J. Pharmacol. 372 (1999), 215-220). Thus, PINwould be an obvious However, due to the pleitrophic action of NO, adeficiency in PIN function would generate many unrelated disordersthroughout the body. Thus, without the information presented in thedisclosure herein, a person of ordinary skills in the art would havepredicted PIN and not P2X7R as the gene associated with affectivedisorders.

The human phospholipase A2 group IB (PLA2G1B) catalyses the release offatty acids from glycero-3-phosphocholines. Phospholipase A2 genes(PLA2) are expressed in many tissues. Some studies have demonstratedassociations between excessive PLA2 activity in brain and affectivedisorders (Chang et al., Neurochem. Res. 23 (1998), 887-892; Hibbeln etal., Biol. Psychiatry 25 (1989), 945-961). Moreover, other geneticstudies have found associations between PLA2G1B gene and bipolaraffective disorder (Dawson et al., Psychiatr. Genet. 5 (1995), 177-180).Thus, PLAG1B represent a likely candidate for affective disorders.However in the present example, only a single silent mutation was foundwithin exon 3 of the PLAG1B gene.

The human citron kinase gene, Rho-associated protein (CIT) is a 183 kDaprotein which associates to the GTPase Rho. CIT shares strong similaritywith ROCK and ROK proteins which are other Rho-associated kinases(Madaule et al., Nature 394 (1998), 491-494). Rho GTPases are involvedin many processes such as cytoskeletal organization, membranetrafficking, cell growth, and transcriptional activation (Van Aelst andD'Souza-Schorey, Genes Dev. 11 (1997), 2295-2322). Studies on brainvariants of Citron-K (without the kinase domain) reveal the associationwith postsynaptic density proteins (PSD-95), suggesting a role in eithersynapse organization or function (Zhang et al., J. Neurosci. 19 (1999),96-108; Furuyashiki et al., J. Neurosci. 19 (1999), 109-118).

The human paxillin (PXN) gene encodes for a 68 kDa protein found infocal adhesions. It is within focal adhesions where adhesion moleculesdynamically interact with the cytoskeleton (Salgia et al., J. Biol.Chem. 270 (1995), 5039-5047). The signaling pathways that regulate thesedynamic interactions begin to be elucidated. Many observations suggestthat paxillin is involved in transducing signals from growth factorreceptors to focal adhesions. The paxillin is expressed in many tissuesincluding brain.

However as set forth below, the gene causative for affective diseases isidentified as being the P2X7 receptor (P2X7R).

Mutations were searched in coding sequences and exon-intron boundariesof the above mentioned genes since such mutations are more likely togive a functionally significant Single Nucleotide Polymorphisms (SNP).The starting sample was composed of 16 unrelated affected individualsfrom the Saguenay/Lac St-Jean region, which gives an 80% power to detectpolymorphisms with a frequency of 0.05. To identify polymorphisms,targeted sequences were first amplified by PCR. Then, PCR products arepurified on Whatman GF/C membranes (VWR, Montreal, Canada), andquantified using the PicoGreen dsDNA quantitation assay (Molecularprobes, Oregon, USA). 4 ng of purified PCR products are sequenced usingthe DYEnamic ET terminator cycle sequencing kit (Amersham Biosciences,Baie D'Urfé, Canada). The sequencing products are resolved on an ABIPRISM 3730XL DNA analyzer, and an ABI PRISM 3700 DNA analyzer. The PCRproducts are sequenced in both directions. The SNPs identified instudied genes are listed in Table 10. TABLE 10 Mutation analysis betweenmarkers D12S1666 and NBG9 Genes Positions Variations AllelesModifications Rab35 Exon06 RABE06A 486G-A Silent Asn162 Rab35 Intron04RABI04A 51C-T unknown Rab35 Intron03 RABI03A 33G-A unknown Rab35Intron02 RABI02B 85G-A unknown Rab35 Intron02 RABI02A 76C-G unknown PXNExon11 PXNE11A 1527C-T Silent Thr509 PXN Exon06 PXNE06A 750C-T SilentSer250 PXN Exon02 PXNE02A 217G-A Gly73Ser PLA2G1B Exon03 PLA2G1BE03A294C-T Silent Ser98 PIN 5′UTR01 PINUTR01A −49T-G unknown PIN 5′UTR01PINUTR01B −80T-C unknown PIN Intron02 PINI02A 26C-T unknown PIN Intron02PINI02B 50C-T unknown CaBP Intron04 CaBPI04A 35C-T unknown CaBP exon01CaBPE01A −23A-G unknown OASL Exon02 OASLE02A 213G-T Silent Gly72 OASLExon02 OASLE02B 408C-T Silent Leu136 OASL Exon05 OASLE05A 1042G-AVal348Met OASL Exon06 OASLE06A 1509G-A Silent Ser503 P2X7R 5′UTRP2XR7UTR5L 362T-C unknown P2X7R 5′UTR P2XR7UTR5M 532T-G unknown P2X7R5′UTR P2XR7UTR5K 1100A-G unknown P2X7R 5′UTR P2XR7UTR5J 1122A-G unknownP2X7R 5′UTR P2XR7UTR51 1171C-G unknown P2X7R 5′UTR P2XR7UTR5F 1351T-Cunknown P2X7R 5′UTR P2XR7UTR5N 1702G-A unknown P2X7R 5′UTR P2XR7UTR5G1731T-G unknown P2X7R 5′UTR P2XR7UTR5H 1860C-T unknown P2X7R 5′UTRP2XR7UTR5A 2162C-A unknown P2X7R 5′UTR P2XR7UTR5B 2238C-T unknown P2X7R5′UTR P2XR7UTR5D 2373A-G unknown P2X7R 5′UTR P2XR7UTR5E 2569G-A unknownP2X7R 5′UTR P2XR7UTR5C 2702G-A unknown P2X7R Intron01 P2XR7I01C 3166G-Cunknown P2X7R Intron01 P2XR7I01A 24778C-T unknown P2X7R Intron01P2XR7I01B 24830C-T unknown P2X7R Exon02 P2XR7v02A 24942T-C Val76AlaP2X7R Exon03 P2XR7E03A 26188C-T Arg117Trp P2X7R Intron03 P2XR7I03A26308A-G unknown P2X7R Intron03 P2XR7I03B 26422G-A unknown P2X7RIntron04 P2XR7I04A 32394G-A unknown P2X7R Intron04 P2XR7v05B 32434T-Cunknown P2X7R Exon05 P2XR7E05D 32493G-A Gly150Arg P2X7R Exon05 P2XR7v05A32507C-T Tyr155His P2X7R Exon05 P2XR7E05C 32783C-T Silent Cys168 P2X7RIntron05 P2XR7I05C 32783A-C unknown P2X7R Intron05 P2XR7I05D 35309T-Cunknown P2X7R Intron05 P2XR7I05B 35374C-T unknown P2X7R Intron05P2XR7I05A 35378A-C unknown P2X7R Exon06 P2XR7E06A 35438G-A Glu186LysP2X7R Exon06 P2XR7E06B 35454T-C Leu191Pro P2X7R Intron06 P2XR7I06C35549T-C unknown P2X7R Intron06 P2XR7I06G 35641G-C unknown P2X7RIntron06 P2XR7I06D 35725A-C unknown P2X7R Intron06 P2XR7I06F 36001T-Gunknown P2X7R Intron06 P2XR7I06E 36064A-T unknown P2X7R Intron06P2XR7I06A 36091DelGTTT unknown P2X7R Intron06 P2XR7I06B 36108C-G unknownP2X7R Intron07 P2XR7I07A 36374C-T unknown P2X7R Intron07 P2XR7I07B36378G-A unknown P2X7R Intron07 P2XR7I07C 36387T-A unknown P2X7RIntron07 P2XR7I07D 36398G-C unknown P2X7R Intron07 P2XR7I07E 37439C-Tunknown P2X7R Intron07 P2XR7I07F 37513T-C unknown P2X7R Exon08 P2XR7E08C37604C-T Arg270Cys P2X7R Exon08 P2XR7v08A 37605G-A Arg270His P2X7RExon08 P2XR7v08B 37623G-A Arg276His P2X7R Exon08 P2XR7E08D 37633C-TSilent Asp279 P2X7R Intron09 P2XR7v11A 47214C-T unknown P2X7R Exon11P2XR7v11B 47383G-A Ala348Thr P2X7R Exon11 P2XR7v11C 47411C-G Thr357SerP2X7R Intron11 P2XR7I11D 47563T-C unknown P2X7R Intron12 P2XR7I12A54307C-T unknown P2X7R Intron12 P2XR7I12B 54308G-A unknown P2X7R Exon13P2XR7v13F 54399C-T Ala433Val P2X7R Exon13 P2XR7v13A 54480A-G Gln460ArgP2X7R Exon13 P2XR7v13B 54523C-T Silent Pro474 P2X7R Exon13 P2XR7v13G54562DelCCCT Del of 7aa 488 GAGAG to 494 CCACAGGTGCCT PESHRCL P2X7RExon13 P2XR7v13C 54588A-C Glu496Ala P2X7R Exon13 P2XR7v13H 54664C-GSilent His521 P2X7R Exon13 P2XR7E13D 54703G-T Silent Leu534 P2X7R Exon13P2XR7E13J 54804A-T Ile568Asn P2X7R Exon13 P2XR7v13I 54834G-A Arg578GlnP2X7R Exon13 P2XR7v13E 54847G-A Silent Pro582 P2X7R 3′UTR P2XR7UTR3A55169C-A unknown P2X7R 3′UTR P2XR7UTR3B 55170A-C unknown P2X7R 3′UTRP2XR7UTR3C 55171A-C unknown P2X7R 3′UTR P2XR7UTR3D 55917C-T unknownP2X7R 3′UTR P2XR7UTR3E 54925G-A unknown P2X4R 5′UTR P2XR4UTR5I −1956G-Aunknown P2X4R 5′UTR P2XR4UTR5H −1649G-A unknown P2X4R 5′UTR P2XR4UTR5G−800G-A unknown P2X4R 5′UTR P2XR4UTR5A −648C-A unknown P2X4R 5′UTRP2XR4UTR5B −537A-G unknown P2X4R 5′UTR P2XR4UTR5C −437A-G unknown P2X4R5′UTR P2XR4UTR5J −206VNRG unknown P2X4R 5′UTR P2XR4UTR5D −211C-G unknownP2X4R 5′UTR P2XR4UTR5F −150VNRGGGCCCC unknown P2X4R 5′UTR P2XR4UTR5E−98G-T unknown P2X4R Intron01 P2XR4I01A 31G-T unknown P2X4R Exon02P2XR4E02A 262G-A Silent mutation Ala87 P2X4R Intron02 P2XR4I02A 4600C-Tunknown P2X4R Intron03 P2XR4I03A 15G-A unknown P2X4R Intron03 P2XR4I03B72G-A unknown P2X4R Exon04 P2XR4E04A 355G-A Ilel19Val P2X4R Exon04P2XR4E04A 375G-A Silent Val125 P2X4R Intron04 P2XR4I04B 17T-C unknownP2X4R Intron04 P2XR4I04A 32G-A unknown P2X4R Exon05 P2XR4E05A 465T-CSilent Ser155 P2X4R Exon07 P2XR4E07A 724A-G Ser242Gly P2X4R Intron05P2XR4I08A DelT unknown P2X4R Exon09 P2XR4E09A 944A-G Tyr315Cys P2X4RIntron10 P2XR4I10A 11G-T unknown P2X4R Intron10 P2XR4I10B G-C unknownP2X4R Intron10 P2XR4I10C A-G unknown P2X4R Intron11 P2XR4I11B C-Gunknown P2X4R Intron11 P2XR4I11C T-A unknown P2X4R Intron11 P2XR4I11A374C-T unknown CaMKK2 3′UTR CaMKK2UTR3bA 733C-T unknown CaMKK2 3′UTRCaMKK2UTR3aB 390G-A unknown CaMKK2 3′UTR CaMKK2UTR3aA 239G-A unknownCaMKK2 Intron15 CaMKK2I15B 325T-C unknown CaMKK2 Intron15 CaMKK2I15A169G-A unknown CaMKK2 Intron14 CaMKK2I14A 224A-G unknown CaMKK2 Intron10CaMKK2I10A 156DelGTGATCC unknown GCCTG CaMKK2 intron09 CaMKK2I09B 528A-Gunknown CaMKK2 intron09 CaMKK2I09A 521A-G unknown CaMKK2 Exon09SNP6f18v5 1095C-A Silent Ile365 CaMKK2 Exon09 SNP6f18v4 1087C-TArg363Cys CaMKK2 Exon05 CaMKKE05A 687C-T Silent Pro229 CaMKK2 Intron03CaMKK2I03A 10C-T unknown CaMKK2 Intron02 CaMKK2I02A 39C-T unknown CaMKK2Intron01 CaMKK2I01B 2911G-C unknown CaMKK2 Intron01 CaMKK2I01A 89C-Aunknown CaMKK2 Exon01 SNP6f18v2 253A-T Thr85Ser CaMKK2 Exon01 SNP6f18v129G-A Ser10Asn CaMKK2 5′UTR01 CaMKK2UTR01B 253T-C unknown CaMKK2 5′UTR01CaMKK2UTR01A 63C-A unknown APC5 Intron01 APC5I01A 10G-T unknown APC5Intron01 APC5I01B 50A-T unknown APC5 Intron05 APC5I05A 73T-C unknownAPC5 Intron06 APC5I06A 73T-G unknown APC5 Exon11 APC5E11A 1416C-T SilentHis472

Each SNP in genes Rab35, PXN, PLA2G1B, PIN, CaBP, OASL, P2X4R, CaMKK2and APC5 was designated according to the gene where it was found, andits location in that gene (intronic or exonic regions). Each SNP in theP2X7R gene was designated according to their position on SEQ ID NO: 1.The allele describes the position and the variation observed. In codingregions, the position is relative to the start codon, whereas theintronic SNPs are positioned relative to the beginning of thecorresponding intron (when known). Primers used for identifying the SNPsin the P2X7R and the location of each SNPs included in tables 2 and 12are defined in table 1a and SEQ ID NOs 52 to 111.

Association studies using missense SNPs were performed. Missense SNPs orSNPs that could be close to the splice sites were used, because it ismore likely that diseases would be associated to an improper function inproteins. Case group was composed by bipolar I individuals,schizoaffective bipolar type (182 subjects) and bipolar II diagnosedpersons (31 subjects). Many controls from the Saguenay/Lac-St-Jeanregion, were sampled from Steinert, Glaucoma and Paget DNA banks. Thecontrol individuals were not diagnosed for affective disorders.According to the lifetime risks of bipolar disorders (1%), there is noneed to screen controls for psychiatric disorders.

Direct sequencing of PCR products is by far the most accurate method ofanalysis and is the method of choice in view of our sequencing platformcapacity. PCR products were analyzed by direct sequencing as describedabove. After sequencing analysis, individuals are automatically typedfor the corresponding SNP using a home-developed program, GENO.pl. Theresults of SNP genotyping are compiled in a 4D database.

The association hypothesis was tested with CLUMP (Sham & Curtis 1995,Ann. Hum. Genet. 59:97-105). One thousand simulations were used toestimate p-values. Results are illustrated in table 11. The T1statistic, which is the usual chi-squared statistic on the rawcontingency table, was used to test for allelic association. Moreover,the largest chi-squared statistic got by comparing one column of theoriginal table against the total of the other columns, called T3statistic, was added to the previous one to test for potential genotypeassociation since T1 statistic results may be biased when thecontingency table contains cells with low values. TABLE 11 Associationhypothesis using CLUMP Genotype Allele Analysis Analysis p- Effectivep-value p-value value gene SNPs Cases Controls (T1) (T2) (T3) P2X7RP2XR7v11B 208 211 0.795 0.036 0.028 P2XR7v13A 212 214 0.344 0.250 0.186P2XR7v13E 212 211 0.780 0.017 0.017 CAMKK2 SNP6f18v5 206 135 1.00 1.001.00 SNP6f18v4 206 135 0.816 0.962 0.841 SNP6f18v2 205 135 0.057 0.1100.095 SNP6f18v1 206 135 0.512 0.532 0.385

The association studies using SNPs in P2X7, P2X4, and CaMKK2 revealassociations significant at level of about 5% or less. Three genotypeassociations in P2X7 were observed. However, SNPs P2XR7v11B andP2XR7v13E are closely linked together based on a contingency table.There is also an allele association at level of 5.7% for SNP6f18v2 inCaMKK2. The information associated to each relevant SNP can be found inTables 10 and 12.

Further association studies using CLUMP were performed on samples thatcontain more case and control individuals. One thousand simulations wereused to estimate p-values. TABLE 11a Empirical p-values and odds ratio(OR) with 95% confidence interval observed with CLUMP for alleles andgenotypes analysis of SNPs Alleles Genotypes T1 OR T1 T3 Marker AlleleEffective p- 95% p- p- Gene (marker rank) Frequencies case controlsvalue OR CI value value P2XR7 P2XR7UTR5F C (0.18); T 212 208 0.280 1.210.86-1.71 0.067 0.069 (1) (0.82) P2XR7UTR5G G (0.09); T 211 204 0.4811.19 0.76-1.87 0.261 0.231 (2) (0.91) P2XR7UTR5H C (0.95); T 210 2020.549 1.19 0.67-2.13 0.768 0.582 (3) (0.05) P2XR7UTR5A A (0.05); C 210207 0.526 1.26 0.68-2.34 0.754 0.517 (4) (0.95) P2XR7UTR5B C (0.78); T211 207 0.629 1.09 0.79-1.50 0.104 0.128 (5) (0.22) P2XR7UTR5D A (0.96);G 211 205 0.268 1.43 0.77-2.65 0.598 0.240 (6) (0.04) P2XR7UTR5E A(0.04); G 211 210 0.658 1.23 0.65-2.33 0.139 0.234 (7) (0.96) P2XR7UTR5CA (0.22); G 208 210 0.889 1.04 0.75-1.44 0.168 0.293 (8) (0.78)P2XR7I01B C (0.98); T 210 207 0.352 1.71 0.67-4.39 0.348 0.348 (9)(0.02) P2XR7v02A C (0.05); T 211 208 0.189 1.49 0.84-2.64 0.397 0.167(10) (0.95) P2XR7I04A A (0.01); G 211 211 0.344 0.25 0.03-2.23 0.3560.356 (11) (0.99) P2XR7v05B C (0.75); T 212 211 0.854 1.03 0.76-1.410.234 0.335 (12) (0.25) P2XR7E05D A (0.01); G 211 211 0.726 1.510.42-5.38 0.735 0.735 (13) (0.99) P2XR7v05A C (0.48); T 211 209 0.6381.07 0.82-1.40 0.895 0.895 (14) (0.52) P2XR7E05C C (0.97); T 210 2110.195 0.45 0.16-1.31 0.349 0.276 (15) (0.03) P2XR7I07E C (0.64); T 208214 0.394 0.87 0.66-1.16 0.057 0.064 (16) (0.36) P2XR7v08A A (0.24); G210 212 0.221 1.22 0.90-1.67 0.433 0.496 (17) (0.76) P2XR7v08B A (0.05);G 210 213 0.386 0.71 0.36-1.41 0.520 0.662 (18) (0.95) P2XR7V11A C(0.88); T 213 149 0.394 0.80 0.50-1.29 0.387 0.463 (19) (0.12) P2XR7v11BA (0.36); G 208 211 0.795 1.04 0.79-1.38 0.036 0.028 (20) (0.64)P2XR7v11C C (0.89); G 211 212 0.409 0.82 0.52-1.28 0.303 0.661 (21)(0.11) P2XR7v13F C (0.99); T 196 207 0.030 3.24 1.04-10.12 0.039 0.039(22) (0.01) P2XR7v13A A (0.84); G 212 214 0.344 1.21 0.85-1.72 0.2500.186 (23) (0.16) P2XR7v13B C (0.89); T 207 212 0.494 0.83 0.53-1.310.315 0.699 (24) (0.11) P2XR7v13C A (0.77); 211 213 0.731 0.95 0.68-1.310.557 0.616 (25) C(0.23) P2XR7V13H C (0.98); G 211 213 0.238 1.750.68-4.49 0.236 0.236 (26) (0.02) P2XR7E13D G (0.89); T 211 213 0.4350.82 0.53-1.28 0.268 0.680 (27) (0.11) P2XR7E13J A (0.03); T 204 1990.179 0.48 0.16-1.42 0.329 0.329 (28) (0.97) P2XR7v13E A (0.36); G 212213 0.841 1.04 0.79-1.37 0.026 0.025 (29) (0.64) P2XR7UTR3E A (0.04); G205 197 1.000 0.96 0.45-2.04 1.000 1.000 (30) (0.96) P2XR7UTR3A A(0.47); C 208 209 0.932 0.99 0.75-1.30 0.264 0.239 (31) (0.53)P2XR7UTR3B A (0.92); C 208 210 0.174 0.65 0.38-1.14 0.151 0.303 (32)(0.08) P2XR7UTR3C A (0.95); C 208 210 0.395 0.71 0.36-1.40 0.508 0.667(33) (0.05) P2XR4 UTR5A A (0.18); C 212 210 0.285 0.82 0.57-1.18 0.5140.484 (0.82) UTR5B A (0.69); G 212 210 0.670 0.93 0.70-1.25 0.833 0.833(0.31) I06A C (0.84); T 207 192 0.212 0.78 0.53-1.16 0.398 0.217 (0.16)E07A A (0.84); G 212 208 0.294 0.81 0.55-1.19 0.536 0.479 (0.16) UTR3A C(0.74); G 211 203 0.015 1.50 1.11-2.02 0.021 0.014 (0.26) UTR3B A(0.97); T 211 209 0.653 0.81 0.33-1.97 0.649 0.649 (0.03) UTR3C C(0.03); G 211 209 0.653 0.81 0.33-1.97 0.672 0.672 (0.97) CAMKK2 E09B A(0.03); C 208 214 0.830 0.85 0.36-2.00 0.829 0.829 (0.97) E09A C (0.83);T 208 214 0.202 0.78 0.54-1.14 0.446 0.473 (0.17) E01B A (0.35); T 207214 0.048 1.33 1.01-1.76 0.126 0.218 (0.65) E01A C (0.93); T 208 2140.189 1.44 0.86-2.39 0.439 0.237 (0.07)

Thirty-three SNPs in P2X7R, seven SNPs in P2X4R, and four SNPs inCAMKK2, with minor allele frequency higher or equal to 1% were genotyped(Table 11a). The genotype distributions of these SNPs did not deviatesignificantly from HWE. At the 5% level, statistically significantincreases of minor allele frequency were observed in the bipolaraffective disorder group at p2XR7v13F (p-value=0.030, OR=3.24, 95%CI=1.04-10.12), P2XR4UTR3A (p-value=0.015, OR=1.50, 95% CI=1.11-2.02)and CAMKK2E01B (p-value=0.048, OR=1.33, 95% CI=1.01-1.76). Thedistribution of genotypes at SNPs P2XR7v13F and P2XR4UTR3A also differedsignificantly at this level for T1 and T3 statistics, with an increaseof heterozygotes in the case sample. One SNP from exon 11 of P2X7R,P2XR7v11B, and another from exon 13, P2XR7v13E, displayed difference ingenotype distributions with minimum p-value of 0.028 and 0.025 observedboth with T3 statistic. Again, increase in heterozygote frequency of 12%and 13% were respectively observed in the bipolar sample at thesepolymorphisms.

Significant haplotypic association tests led to p-values less than 0.5%for different SNP groups overlapping the P2X7R gene (Table 11b).Considering the SNPs collection ranging from SNP32507 to SNP54847 (table11c) as an example for haplotype distribution, we observed the largestdifference of frequencies between cases and controls with the haplotypeno 1 (table 11 d). The haplotype no 2 is another example of haplotypethat is more frequently observed in cases group. On the other hand, thefrequency for haplotype no 3 is slightly increased in control sample(difference of frequencies=0.091). Table 11e presents the peptidicproducts derived from the nucleotidic haplotypes shown in table 11d.TABLE 11b Haplotypes showing allelic association significant at the 0.5%level for T1 or T3 statistics. Haplotype T1 Haplotype Distance²effective statistic T3 statistic (marker ranks¹) #SNPs (bp) case control(p-value) (p-value) #haplotype³ P2XR7I01B-P2XR7v13A 15 29618 361 2570.0003 0.0252 20 (9-23) P2XR7v02A-P2XR7v13B 15 29550 361 260 0.000080.0294 20 (10-24) P2XR7I04A-P2XR7v13C 15 22164 360 264 0.0003 0.0323 19(11-25) P2XR7v05B-P2XR7v13H 15 22200 361 265 0.0004 0.0065 18 (12-26)P2XR7E05D- 15 22180 365 268 0.0035 0.0287 16 P2XR7E13D (13-27)P2XR7v05A-P2XR7E13J 15 22267 352 246 0.0007 0.0163 15 (14-28) P2XR7E05C-15 22269 353 250 0.0012 0.0200 10 P2XR7v13E (15-29) P2XR7I07E- 15 17452355 247 0.0020 0.0192 11 P2XR7UTR3E (16-30)¹The marker ranks of SNPs in the haplotype indicated in table 11b referto those genotyped in table 11a²Distance between the two most distal SNPs of the haplotype³Number of haplotypes with frequencies >1% in case or control groups.

TABLE 11c Position and Allele for haplotype-forming SNPs. Haplotypes aredescribed in table 11d. SEQ ID NO Polymorphism Position 1 C-T 32507 1C-T 32548 1 C-T 37439 1 G-A 37605 1 G-A 37623 1 C-T 47214 1 G-A 47383 1C-G 47411 1 C-T 54399 1 A-G 54480 1 C-T 54523 1 A-C 54588 1 C-T 54664 1G-T 54703 1 T-A 54804 1 G-A 54847

TABLE 11d haplotypes with significant difference of frequencies betweenaffected and control individuals. Haplotype 32507 32548 37439 3760537623 47214 47383 47411 54399 54480 No 1 C C C A G C G C C A No 2 C C CG G C G C C A No 3 C C T G G C A C C A Haplotype 54523 54588 54664 5470354804 54847 F_(affected) F_(controls) No 1 C A C G T G 0.20 0.13 No 2 CC C G T G 0.05 0.01 No 3 C A C G T A 0.11 0.20

TABLE 11e Corresponding amino acids for cSNPs described in table 11c.They are positioned according to SEQ ID NO3. Position in SEQ ID NO3 155168 270 276 348 357 433 Haplotype 1 Y C H R A T A Haplotype 2 Y C R R AT A Haplotype 3 Y C R R T T A Position in SEQ ID NO3 460 474 496 521 534568 582 Haplotype 1 Q P E H L I P Haplotype 2 Q P A H L I P Haplotype 3Q P E H L I P

EXAMPLE 3 Polymorphisms Found in the P2X7R in Individuals Suffering fromDepression

Association studies using SNPs in the P2X7R gene was performed in acase/control sample (535 individuals) from a German population. The casegroup was composed of 36 individuals diagnosed with bipolar type I ortype II, and 279 individuals diagnosed with unipolar disorders (i.e.depression) representing 133 affected males and 182 affected females.Among controls, we count The remaining 220 control individuals werenormal (i.e. diagnosed as non depressive), and comprising 81 males, 182females and 14 of unknown gender. The same sexual distribution was notedin both groups.

SNPs were identified in this sample by using a subgroup of 24 affectedindividuals. SNPs in the P2X7R gene detected in the German populationwere similar if not identical to the SNPs seen in theSaguenay/Lac-St-Jean population (see table 12). Other rare missense SNPswere also noted in the German population, such as Arg117Trp (P2XR7E03A),Glu186Lys (P2XR7E06A), Leu191Pro (P2XR7E06B), Ile568Asn (P2XR7E13J).These amino acids are quite conserved between ortholog P2X7 genes. It ispossible that the Ile568Asn (P2XR7E13J) mutation may be involved in thesurface expression of P2X7. TABLE 12 Comparison between polymorphisms inthe Saguenay/Lac-St-Jean population and the German population in thehuman P2X7R gene Associated Variation exons or (SNP Frequency Frequencyintrons or others) Allele Position* Modification (Canada) (Germany)5′UTR P2XR7UTR5L T-C 362 unknown 0.13 0.08 5′UTR P2XR7UTR5M T-G 532unknown 0.16 0.1 5′UTR P2XR7UTR5K A-G 1100 unknown 0.13 0.13 5′UTRP2XR7UTR5J A-G 1122 unknown 0.13 0.13 5′UTR P2XR7UTR5I C-G 1171 unknown0.06 0.02 5′UTR P2XR7UTR5F T-C 1351 unknown 0.3 0.12 5′UTR P2XR7UTR5NG-A 1702 unknown — 0.02 5′UTR P2XR7UTR5G T-G 1731 unknown 0.17 0.155′UTR P2XR7UTR5H C-T 1860 unknown 0.07 0.15 5′UTR P2XR7UTR5A C-A 2162unknown 0.07 0.12 5′UTR P2XR7UTR5B C-T 2238 unknown 0.3 0.27 5′UTRP2XR7UTR5D A-G 2373 unknown 0.07 0.12 5′UTR P2XR7UTR5E G-A 2569 unknown0.1 0.02 5′UTR P2XR7UTR5C G-A 2702 unknown 0.31 0.27 Intron01 P2XR7I01CG-C 3166 unknown 0.03 — Intron01 P2XR7I01A C-T 24778 unknown 0.03 —Intron01 P2XR7I01B C-T 24830 unknown 0.03 RARE Exon02 P2XR7v02A T-C24942 Val76Ala 0.06 0.08 Exon03 P2XR7E03A C-T 26188 Arg117Trp — RAREIntron03 P2XR7I03A A-G 26308 unknown 0.7 0.44 Intron03 P2XR7I03B G-A26422 unknown 0.18 0.12 Intron04 P2XR7I04A G-A 32394 unknown 0.03 0.01Intron04 P2XR7v05B T-C 32434 unknown 0.33 0.29 Exon05 P2XR7E05D G-A32493 Gly150Arg RARE 0.02 Exon05 P2XR7E05E G-A 32506 Silent — RAREVal154 Exon05 P2XR7v05A C-T 32507 Tyr155His 0.33 0.44 Exon05 P2XR7E05CC-T 32548 Silent RARE 0.02 Cys168 Intron05 P2XR7I05C A-C 32783 unknown0.25 — Intron05 P2XR7I05D T-C 35309 unknown ND 0.35 Intron05 P2XR7I05BC-T 35374 unknown 0.7 0.67 Intron05 P2XR7I05A A-C 35378 unknown 0.7 0.65Exon06 P2XR7E06A G-A 35438 Glu186Lys — 0.02 Exon06 P2XR7E06B T-C 35454Leu191Pro — 0.02 Intron06 P2XR7I06C T-C 35549 unknown 0.04 0.08 Intron06P2XR7I06G G-C 35641 unknown — 0.02 Intron06 P2XR7I06D A-C 35725 unknown0.21 0.27 Intron06 P2XR7I06F T-G 36001 unknown 0.17 0.3 Intron06P2XR7I06E A-T 36064 unknown 0.11 0.1 Intron06 P2XR7I06A DelGTTT36091-36094 unknown 0.14 0.3 Intron06 P2XR7I06B C-G 36108 unknown 0.140.29 Intron07 P2XR7I07A C-T 36374 unknown 0.07 — Intron07 P2XR7I07B G-A36378 unknown 0.21 0.28 Intron07 P2XR7I07C T-A 36387 unknown 0.21 0.28Intron07 P2XR7I07D G-C 36398 unknown 0.42 0.4 Intron07 P2XR7I07E C-T37439 unknown 0.41 — Intron07 P2XR7I07F T-C 37513 unknown — RARE Exon08P2XR7E08C C-T 37604 Arg270Cys RARE — Exon08 P2XR7v08A G-A 37605Arg270His 0.46 0.24 Exon08 P2XR7v08B G-A 37623 Arg276His 0.03 0.02Exon08 P2XR7E08D C-T 37633 Silent RARE — Asp279 Intron09 P2XR7v11A C-T47214 unknown 0.08 0.03 Exon11 P2XR7v11B G-A 47383 Ala348Thr 0.5 0.44Exon11 P2XR7v11C C-G 47411 Thr357Ser 0.08 0.07 Intron11 P2XR7I11D T-C47563 unknown 0.43 0.44 Intron12 P2XR7I12A C-T 54307 unknown 0.32 —Intron12 P2XR7I12B G-A 54308 unknown 0.03 — Exon13 P2XR7v13F C-T 54399Ala433Val 0.13 — Exon13 P2XR7v13A A-G 54480 Gln460Arg 0.13 0.17 Exon13P2XR7v13B C-T 54523 Silent 0.1 0.07 Pro474 Exon13 P2XR7v13G DelCCCTGAGA54562-54582 Del of 7aa RARE — GCCACAGG 488 to 494 TGCCT (PESHRCL) Exon13P2XR7v13C A-C 54588 Glu496Ala 0.13 0.06 Exon13 P2XR7v13H C-G 54664His521Gln 0.03 — Exon13 P2XR7E13D G-T 54703 Silent 0.1 0.02 Leu534Exon13 P2XR7E13J A-T 54804 Ile568Asn — 0.01 Exon13 P2XR7v13I G-A 54834Arg578Gln — RARE Exon13 P2XR7v13E G-A 54847 Silent 0.4 0.45 Pro582 3′UTRP2XR7UTR3A C-A 55169 unknown 0.48 0.37 3′UTR P2XR7UTR3B A-C 55170unknown 0.09 0.1 3′UTR P2XR7UTR3C A-C 55171 unknown 0.05 0.06 3′UTRP2XR7UTR3D C-T 55917 unknown 0.001 — 3′UTR P2XR7UTR3E G-A 54925 unknown— 0.01

The position and numbering of the polymorphism corresponds to the humanP2X7R gene as defined in SEQ ID NO: 1. To identify the genomicorganization of the P2X7R gene, BAC clones were firstly organized usingknown polymorphic markers, sequence tag sites (STSs), BAC-end sequencesand expressed sequence tags (ESTs). Unorientated and unordered DNAregions were reassembled into a sequences using Phrap and reordered thepieces using P2X7R exons as scaffolds. No complete gene organization forP2X7R has been done. There is only a partial gene structure from exon6to 13, NT_(—)037809. Therefore, this genomic sequence encompassing theP2X7R gene as depicted in SEQ ID NO: 1 could contain some sequenceerrors, specifically in intronic regions. Primers used for SNPamplification and sequencing are shown in Table 1a and depicted in SEQID NOs: 52 to 111.

Statistical analysis was performed according to the CLUMP method (Sham &Curtis 1995, Ann. Hum. Genet. 59:97-105). Table 13 resumes the allelicand genotypic association studies for SNPs in P2X7 gene. TABLE 13Allelic and genotypic association studies using CLUMP Allele EffectiveAnalysis Genotype Analysis Locus Allele Frequencies* Cases Controlsp-value (T1) p-value (T1) p-value (T3) P2XR7UTR5F 2(0.23); 4(0.77) 311217 0.109 0.319 0.339 P2XR7UTR5N 1(0.001); 3(0.999)** 314 218 0.0380.048 0.048 P2XR7UTR5G 2(0.001); 3(0.105); 4(0.894) 314 218 0.993 0.7140.761 P2XR7UTR5H 2(0.92); 4(0.08) 312 215 0.743 0.884 0.754 P2XR7UTR5A1(0.08); 2(0.92) 312 219 0.557 0.786 0.678 P2XR7UTR5B 2(0.73); 4(0.27)310 218 0.485 0.761 0.814 P2XR7UTR5D 1(0.92); 3(0.08) 311 217 0.5550.787 0.691 P2XR7v02A 2(0.09); 4(0.91) 313 218 0.501 0.729 0.591P2XR7I04A 1(0.04); 3(0.96) 314 220 0.604 0.433 0.348 P2XR7v05B 2(0.69);4(0.31) 314 220 0.133 0.270 0.325 P2XR7E05D 1(0.03); 3(0.97) 314 2200.842 0.827 0.827 P2XR7E05E 1(0.006); 3(0.994)** 314 220 0.048 0.0450.045 P2XR7v05A 2(0.60); 4(0.40) 314 220 0.038 0.144 0.219 P2XR7E05C2(0.98); 4(0.02) 314 220 1.000 1.000 1.000 P2XR7I07F 2(0.002); 4(0.98)315 219 1.000 1.000 1.000 P2XR7v08A 1(0.23); 3(0.77) 315 219 0.454 0.6730.634 P2XR7v08B 1(0.02); 3(0.98) 315 219 0.636 0.638 0.638 P2XR7v11A2(0.95); 4(0.05) 311 218 0.348 0.391 0.436 P2XR7v11B 1(0.45); 3(0.55)312 218 0.605 0.803 0.790 P2XR7v11C 2(0.93); 3(0.07) 312 218 0.793 0.2560.924 P2XR7I11D 2(0.45); 4(0.55) 312 219 0.665 0.735 0.740 P2XR7v13A1(0.87); 3(0.13) 305 215 0.017 <0.001 <0.001 P2XR7v13B 2(0.93); 4(0.07)305 216 1.000 0.228 0.677 P2XR7V13C 1(0.91); 2(0.09) 305 216 0.151 0.0060.008 P2XR7E13D 3(0.94); 4(0.06) 315 219 0.402 0.429 0.474 P2XR7E13J1(0.01); 4(0.99) 315 219 0.618 0.603 0.603 P2XR7E13I 1(0.004); 3(0.996)315 219 0.999 1.000 1.000 P2XR7v13E 1(0.46); 3(0.54) 314 219 0.699 0.8660.845 P2XR7UTR3A 1(0.518); 2(0.482) 314 219 0.617 0.850 0.875 P2XR7UTR3B1(0.966); 2(0.034) 313 219 0.522 0.850 0.643 P2XR7UTR3C 1(0.979);2(0.021) 313 219 0.636 0.505 0.382 P2XR7UTR3E 1(0.02); 3(0.98) 315 2190.147 0.161 0.161*The column Allele Frequencies presents the allele for each SNP (A = 1,C = 2, G = 3, T = 4) and their respective frequency.**For this SNP we observed a zero cell in both (allele and genotype) 2 ×2 contingency tables.p-value <0.045 was observed exact Fisher test.

For the SNP analysis, the Hardy-Weinberg (HW) equilibrium was controlledin the control samples. The Hardy-Weinberg principle (HWP) may be statedas follow: In a large, randomly mating population, in which there is nomigration, or selection against a particular genotype and the mutationrate remains constant, the proportions of the various genotypes willremain unchanged from one generation to another. Take a two allelesystem with alleles A and a. If the proportion of A in the population isrepresented as p and the proportion of a as q, then p plus q representthe sum total of alleles at this locus, that is p+q=1. The HWP is usefulto evaluate some population problems like marital assortment,Inbreeding, population stratification, admixture, decreased viability ofa particular genotype. The SNP P2XR7v13A did not respect theHardy-Weinberg equilibrium.

The association hypothesis was also tested using an allele positivitytable known to be suitable for the detection of susceptibility allelesshowing a dominant mode of inheritance (Ohashi and Tokunaga, J. Hum.Genet. 44 (1999), 246-248; Ohashi et al., Ann. Hum. Genet. 65 (2001),197-206). Similar results were obtained using this method as thoseobtained using the allele frequency tables, with the exception ofP2XR7v05A where the p-values were 0.253. Thus, P2XR7v05A presented aless significant association in this analysis. This difference can beattributed to the mode of inheritance.

The proportion of unipolar individuals in analysis of the Germanpopulation is quite important since the American Psychiatric Association(Diagnostic and Statistical Manual of Mental Disorders-4th Edition TextRevision (DMS-IV-TR), American Psychiatric Press, 2000) has reported anincrease in susceptibility for unipolar disorders in female groups. Todetermine whether the sexual variable could influence the associationanalysis, additional association studies were performed by controllingthe sexual parameter. Normal individuals in the German populationwithout gender information were omitted from the study. Then, a logisticregression model was derived by including the sex as factor. In order toobtain a model that is as stable as possible, the regression model wasminimised by using the difference between log-likelihood's for modelswith or without interaction (Hosmer, and Lemeshow, “Applied logisticregression”, John Wiley and Sons, 1989). The strategy used for handlingthe zero cells from contingency tables was to eliminate associatedcategory completely. Calculations were done with SAS v8.0 SAS is astatistical software package that allows the user to manipulate andanalyze data in many different ways. Because of its capabilities, thissoftware package is used in many disciplines, including medicalsciences, biological sciences, and social sciences.

The introduction of a sexual parameter did not perturb the associationalready observed in previous analysis. Moreover, this analysis modelrevealed additional results: a potential allele association withP2XR7v05B (p=0.064), and a genotypic association for P2XR7v08A (p=0.042)was observed.

Association studies using pooled samples was performed by mergingindividuals from the samples of the Saguenay/Lac St-Jean with those ofthe German population. Results are illustrated in table 14. The aim ofthis analysis is to highlight common features between both populations.However, according to differences between both samples (mainly thephenotype of affected individuals i.e. bipolar disorder in theSaguenay/Lac St-Jean samples, versus mostly unipolar disorder in theGerman population) some parameters were controlled, including sex andethnicity. The modelling strategy for logistic regressions was describedabove. TABLE 14 Association studies using pooled samples from bothpopulations Allele analysis Genotype analysis p-value p-value Locus forSNP p-value for sex for SNP p-value for sex P2XR7v02A 0.8254 0.00850.8650 0.4531 P2XR7v05B 0.1751 0.3714 0.2034 0.5110 P2XR7v05A 0.38080.0266 0.0885 0.1392 P2XR7v08A 0.0452 0.0041 0.1021 0.3452 P2XR7v08B0.3471 0.0040 0.3413 0.3617 P2XR7v11A 0.3559 0.0136 0.5888 0.4404P2XR7v11B 0.5902 0.0093 0.3897 0.4302 P2XR7v11C 0.3731 0.0094 0.76480.4615 P2XR7v13A 0.0047 0.0209 <0.0001 0.4814 P2XR7v13B 0.5129 0.23520.9584 0.4092 P2XR7v13C 0.2466 0.0284 0.2225 0.4228 P2XR7v13E 0.81680.0159 0.3713 0.4990

An allelic and genotypic association was observed for the P2XR7v13Alocus (p=0.0047) which was stronger than in the separate analyses. Asignificant allelic association was also noted for the P2XR7v08A locus(p=0.0452). In addition, the present analysis also demonstrate thepotential relationship between SNP P2XR7v05A and the origin with ap-value=0.0515 (not shown in the table) which is in agreement withprevious association analysis done in both samples separately (see Table13).

The haplotype analysis was performed using the German population. ThePHASE program (Stephens et al., Am. J. Hum. Genet. 68 (2001), 978-989)was used to estimate SNPs haplotypes within exons of the P2X7R gene.Haplotypes were created for each exon having more than one associatedSNP (see Table 15 for exon-associated SNPs). Case groups varied from218-220 individuals, whereas control groups varied between 312-316individuals. Association hypothesis was tested with the CLUMP methodsince many haplotypes were created for each exon. T1 and T3 statistictests performed as described above. T2 and T4 statistics were alsocalculated owing to the presence of small effective cells in thecontingency tables. T2 statistic is the usual chi-squared statisticapplied on the contingency table obtained after collapsing columns withsmall expected values. T4 statistic is the largest chi-squared statisticobtained by comparing one column of the original table against the totalof the other columns. One thousand simulations were used to estimatep-values. The resulting data was analyzed with the logistic regressionmodel (describe above) using SAS V8.0 in order to consider der thesexual parameter (for these tests the sample was reduced by 14 normalindividuals). However, this analysis method is limited by thereliability of reconstructed haplotypes. TABLE 15 Exon-associated SNPsExons Associated SNPs 5 P2XR7E05D P2XR7E05E P2XR7v05A P2XR7E05C 8P2XR7v08A P2XR7v08B 11 P2XRv11B P2XRv11C 13 P2XR7v13A P2XR7v13BP2XR7v13C P2XR7E13D P2XR7E13J P2XR7v13I P2XR7v13E

TABLE 16 Genotypic association with haplotypes in exon 13 of P2X7R ExonAllele analysis Genotype analysis (haplotype) Clump* p-value(sex)p-value(haplo) Clump p-value(sex) p-value(haplo)  5(5) T1: 0.032 0.31330.1947 T1: 0.193 0.460 0.5355 T2: 0.068 T2: 0.159 T3: 0.054 T3: 0.099T4: 0.059 T4: 0.304  8(3) T1: 0.551 0.3813 0.3064 T1: 0.812 0.54280.6652 T2: 0.585 T2: 0.689 T3: 0.646 T3: 0.644 T4: 0.646 T4: 0.756 11(3)T1: 0.750 0.0886 0.7396 T1: 0.625 0.2305 0.9494 T2: 0.786 T2: 0.919 T3:0.726 T3: 0.929 T4: 0.726 T4: 0.921 13(15**) T1: 0.088 0.1871 0.1264 T1:0.001 0.4610 0.019 T2: 0.079 T2: 0.002 T3: 0.147 T3: 0.057 T4: 0.072 T4:<0.001*T1 test should not be consider ed because of contingency tables withzero cells.**Among these 15 haplotypes, we observed 8 haplotypes where case cellshave less than 3 individuals.

Table 16 illustrates a genotypic association with haplotypes in exon 13of the P2X7R genes. Interestingly, many haplotypes for the exon 13 wereobserved. The differences between statistics in exon 13 (T3 lesssignificant) can be explained by the involvement of more than onegenotype of haplotypes in the disease. A potential allelic associationwas also noted with haplotypes in exon 5 of the P2X7R gene.

The following are clinical results illustrating the functionalconsequences of polymorphisms in P2X7R.

The development and course of depression is causally linked toimpairments in the central regulation of thehypothalamic-pituitary-adrenocortical (HPA) axis. Abnormalities in theHPA axis can be measured using the dexamethasone-suppression test (DST)or the combined dexamethasone/corticotropin-releasing hormone (Dex/CRH)test. Changes in cortisol and/or adrenocorticotropic hormone (ACTH)measurements during the DST or Dex/CHR test are indicative of HPAdysfunction in depressed patients (Heuser et al, J. Psychiat. Res. 28(1994) 341-356; Rybakowski and Twardowska, J. Psychiat. Res. 33 (1999)363-370; Zobel et al, J. Psychiat. Res. 35 (2001) 83-94; Künzel et al,Neuropsychopharmacology 28 (2003) 2169-2178). In order to demonstratethat P2X7R SNPs associated with affective disorders also correlate withchanges in the HPA axis, cortisol and ATCH levels in response to the DSTand Dex/CRH text were measured for the P2XR7v13A and P2XR7v13C SNPs.P2XR7v13A consist of an A to G nucleotide change resulting in aGln460Arg modification in the P2X7R protein. The P2XR7v13C SNPcorresponds to an A to C nucleotide change resulting in a Glu496Alamodification that has been shown to drastically reduce protein activity(Wiley et al, Drug Dev. Res. 53 (2001) 72-76).

Methods and conditions for performing the DST and Dex/CRH test are wellknown in the art, see for example Heuser et al, J. Psychiat. Res. 28(1994) 341-356; Künzel et al, Neuropsychopharmacology 28 (2003)2169-2178. Briefly, individuals were pre-treated at 23:00 with an oraladministration of 1.5 mg dexamethasone. For the DST test, a blood samplewas drawn at 8:00 prior to to dexamethasone administration (i.e.pre-dexamethasone) and at 8:00 the morning following dexamethasoneadministration (i.e. post-dexamethasone). For the Dex/CRH test, a venouscatheter was inserted at 14:30 the day following dexamethasoneadministration and blood was collected at 15:00, 15:30, 15:45, 16:00,and 16:15 into tubes containing EDTA and trasylol (Bayer Inc., Germany).At 15:02, 100 mg of human CRH (Ferring Inc., Germany) was administeredintravenously. Measurement of plasma cortisol concentrations was doneusing a commercial radioimmunoassay kit (ICN Biomedicals, USA) whileplasma ACTH concentrations was measured using a commercial immunometricassay (Nichols Institute, USA). Both assays were performed according tothe manufacturer specifications.

For the P2XR7v13A SNP, a decrease in basal cortisol levels was seen atadmission in individuals with an AG or GG allele when compared toindividuals with the M allele (FIG. 1 f). During the Dex/CRH test, areduction in cortisol and ATCH response was measured in individuals withthe GG allele when compared to individuals with an AA or AG allele(FIGS. 1 g and 1 h).

Furthermore, response to antidepressant treatment was delayed in GGindividuals (FIG. 1 i).

For the P2XR7v13C SNP, an increase in basal cortisol levels was measuredpost-dexamethasone administration (FIG. 1 j). During the Dex/CRH test,individuals with the CC allele displayed elevated cortisol response(FIG. 1 k), but reduced ATCH response (FIG. 1 l) when compared to AA andAC individuals. These results are indicative of mysregulation of the HPAaxis.

Thus, SNPs in P2X7R correlate with dysfunction in the HPA axis anddemonstrate the functional and clinical consequences of polymorphisms inP2X7R.

EXAMPLE 4 P2X7R Gene Structure and mRNA Expression and TranscriptSequence

A 1700 bp nucleotide sequence corresponding to the human P2X7R promoterwas analyzed by using Matinspector V2.2 and Transfac 4.0 algorithms.This analysis showed that the P2X7R gene does not contain a standardTATA box, but has SP1 sites that can make up for transcriptionalinitiation. Besides the SP1 sequences, there are binding sites for thetranscription factors GATA, Oct and Ikarus. These sites are thought toprovide tissue specificity. Interestingly, the P2X7R promoter hasbinding sites that suggest responsiveness to different cytokines such asAP-1, NFAT and CEBPB.

P2X7R possesses 13 exons and 12 introns (Buell et al., ReceptorsChannels 5 (1998), 347), providing a basis for alternative splicing thatwould yield in theory different transcripts and produce differentisoforms with possible different functions. No alternatively splicedvariant was clearly identified. However, experiments of EST clusteringallowed the description of three splicing variants. One is defined bythe lack of the exon 5. This P2X7v02 variant corresponds to the cloneIMAGE: 3628076 isolated from brain-derived cell lines. The P2X7v02lacking the exon 5 produces a frame shift, thus generating a shorterpolypeptide. The second splicing variant, P2X7v03, is characterized bythe presence of the short intron 10 into the mRNA. This variant issupported by two high quality sequences, the cDNA clone BRAMY2008977 (ACnumber: AK090866) from human amygdala and the EST clone dbEST:7339877derived from an unknown human tumor. The last variant, P2X7v04, isdefined by the lack of the first exon that suggests an alternativepromoter usage closed to the exon 2. A high quality EST clonedbEST:4782844 derived form a head and neck tumor supports this variant.These variants are shown in FIGS. 16 a to 16 e. P2X7 variants. P2X7v01MPACCSCSDVFQYETNKVTRIQSMNYGTIKWFFHVIIFSYVCFALVSDKLYQRKEPVISS P2X7v04MPPVD--------------------------AFPCLPFS---FALVSDKLYQRKEPVISS P2X7v02MPACCSCSDVFQYETNKVTRIQSMNYGTIKWFFHVIIFSYVCFALVSDKLYQRKEPVISS P2X7v03MPACCSCSDVFQYETNKVTRIQSMNYGTIKWFFHVIIFSYVCFALVSDKLYQRKEPVISS1........10........20........30........40........50 P2X7v01VHTKVKGIAEVKEEIVENGVKKLVHSVFDTADYTFPLQGNSFFVMTNFLKTEGQEQRLCP P2X7v04VHTKVKGIAEVKEEIVENGVKKLVHSVFDTADYTFPLQGNSFFVMTNFLKTEGQEQRLCP P2X7v02VHTKVKGIAEVKEEIVENGVKKLVHSVFDTADYTFPLQGNSFFVMTNFLKTEGQEQRLCP P2X7v03VHTKVKGIAEVKEEIVENGVKKLVHSVFDTADYTFPLQGNSFFVMTNFLKTEGQEQRLCP61.......70........80........90........100.......110 P2X7v01EYPTRRTLCSSDRGCKKGWMDPQSKGIQTGRCVVHEGNQKTCEVSAWCPIEAVEEAPRPA P2X7v04EYPTRRTLCSSDRGCKKGWMDPQSKGIQTGRCVVHEGNQKTCEVSAWCPIEAVEEAPRPA P2X7v02EYPTRRTLCSSDRGCKKGWMDPQSKGLLS------------------------------- P2X7v03EYPTRRTLCSSDRGCKKGWMDPQSKGIQTGRCVVHEGNQKTCEVSAWCPIEAVEEAPRPA121......130.......140.......150.......160.......170 P2X7v01LLNSAENFTVLIKNNIDFPGHNYTTRNILPGLNITCTFHKTQNPQCPIFRLGDIFRETGD P2X7v04LLNSAENFTVLIKNNIDFPGHNYTTRNILPGLNITCTFHKTQNPQCPIFRLGDIFRETGD P2X7v02------------------------------------------------------------ P2X7v03LLNSAENFTVLIKNNIDFPGHNYTTRNILPGLNITCTFHKTQNPQCPIFRLGDIFRETGD181......190.......200.......210.......220.......230 P2X7v01NFSDVAIQGGIMGIEIYWDCNLDRWFHHCHPKYSFRRLDDKTTNVSLYPGYNFRYAKYYK P2X7v04NFSDVAIQGGIMGIEIYWDCNLDRWFHHCHPKYSFRRLDDKTTNVSLYPGYNFRYAKYYK P2X7v02------------------------------------------------------------ P2X7v03NFSDVAIQGGIMGIEIYWDCNLDRWFHHCHPKYSFRRLDDKTTNVSLYPGYNFRYAKYYK241......250.......260.......270.......280.......290 P2X7v01ENNVEKRTLIKVFGIRFDILVFGTGGKFDIIQLVVYIGSTLSYFGLAAVFIDFLIDTYSS P2X7v04ENNVEKRTLIKVFGIRFDILVFGTGGKFDIIQLVVYIGSTLSYFGLAAVFIDFLIDTYSS P2X7v02------------------------------------------------------------ P2X7v03ENNVEKRTLIKVFGIRFDILVFGTGGKFDIIQLVVYIGSTLSYFGLVRDSLFHALGKWFG301......310.......320.......330.......340.......350 P2X7v01NCCRSHIYPWCKCCQPCVVNEYYYRKKCESIVEPKPTLKYVSFVDESHIRMVNQQLLGRS P2X7v04NCCRSHIYPWCKCCQPCVVNEYYYRKKCESIVEPKPTLKYVSFVDESHIRMVNQQLLGRS P2X7v02------------------------------------------------------------ P2X7v03EGSD--------------------------------------------------------361......370.......380.......390.......400.......410 P2X7v01LQDVKGQEVPRPAMDFTDLSRLPLALHDTPPIPGQPEEIQLLRKEATPRSRDSPVWCQCG P2X7v04LQDVKGQEVPRPAMDFTDLSRLPLALHDTPPIPGQPEEIQLLRKEATPRSRDSPVWCQCG P2X7v02------------------------------------------------------------ P2X7v03------------------------------------------------------------421......430.......440.......450.......460.......470 P2X7v01SCLPSQLPESHRCLEELCCRKKPGACITTSELFRKLVLSRHVLQFLLLYQEPLLALDVDS P2X7v04SCLPSQLPESHRCLEELCCRKKPGACITTSELFRKLVLSRHVLQFLLLYQEPLLALDVDS P2X7v02------------------------------------------------------------ P2X7v03------------------------------------------------------------481......490.......500.......510.......520.......530 P2X7v01TNSRLRHCAYRCYATWRFGSQDMADFAILPSCCRWRIRKEFPKSEGQYSGFKSPY P2X7v04TNSRLRHCAYRCYATWRFGSQDMADFAILPSCCRWRIRKEFPKSEGQYSGFKSPY P2X7v02------------------------------------------------------- P2X7v03-------------------------------------------------------541......550.......560.......570.......580.......590

Therefore the transcriptional and translational start sequences of thehuman P2X7R were analyzed using Blast, Genescan and HMMgene computersoftware. This analysis indicated that P2X7R possesses with highprobability only one translation start site. Most P2X7R expressionsequence tags (ESTs; Unique cluster Hs. 193470) having a reliable 5′ endshowed identical transcriptional start site. None of the ESTs showed anyindication of alternative splicing. Therefore, in silico analysissuggests that there is a low probability to find different transcriptsproduced by alternative splicing or alternative promoter usage.

The above mentioned in silico data were confirmed by RT-PCR analysisspanning the whole predicted human P2X7R coding sequence using 14 and 19bases (5′-ATGCCGGCTTGCTG-3′; 5′-GTAGGGATACTTGAAGCCA-3′) oligonucleotidescorresponding to the beginning and end of the coding sequence,respectively. Total RNA from whole brain, different dissected brainareas, thymus, spleen and kidney were isolated and analyzed for P2X7Rexpression. RT-PCR reactions were performed using the C. Therm One Steppolymerase system (Roche Applied Science) and a protocol for touch downPCR with hot start. Briefly, Reverse Transcription was performed at 52°C. according to the manufacturer's conditions. PCR reactions wereexecuted with an annealing temperatures of 64° C. for the first fivecycles and of 54° C. for the next 30 cycles.

A single specific band of the size of 1785 bp corresponding to thecomplete coding sequence of P2X7R was detected. P2X7R mRNA was detectedin the whole brain, hippocampus, cerebellum, leukocytes and thymus butnot in cerebral cortex, hypothalamus, spleen and kidney (FIG. 2). AllPCR products were cloned using the pGEM-T-Easy plasmid (Promega),selected in Top-10 bacteria (Invitrogen) by blue-white selection andtested by EcoRI digestion. Clones having fragments of the expected sizewere amplified and purified for sequencing. The sequence confirmed theidentity of the 1785 bp clones as the complete coding sequence ofwild-type P2X7R. Therefore, in all the tissues tested, wild-type P2X7Ris expressed as a single transcript which includes the complete codingsequence. The presence of tissue specific isoforms is unlikely. Thesestudies provide useful information about the P2X7R mRNA expression andtranscript processing. This information can be used to synthesizeriboprobes for in situ hybridization, Northern and Southern blot as wellas engineering cells for the overexpression of P2X7R.

EXAMPLE 5 P2X7R Expression in the Mouse Brain

The expression of P2X7R was further studied by immunohistochemistry ofserial sections of complete mouse brains using a polyclonal antibodydirected against an internal peptide of P2X7R (Santa CruzBiotechnology). The brains from stress-free mice were shock frozen, cutinto 16 μm slices and fixed with paraformaldehide for 5 minutes. Thesections were blocked for 30 minutes at room temperature with 1:10 horseserum. All antibodies were diluted in TBST buffer (Tris-buffered salinewith 0.05% Tween-20). The first antibody was used in a dilution 1:200and incubated overnight. All washes were performed with TBST buffer. Asa secondary antibody, an anti-goat IgG biotinylated (VectorLaboratories) was used and detection was performed using thestreptavidin-biotin-horse-radish peroxidase complex system (VectorLaboratories) in combination with diaminobencidine. Slides werecounterstained with toluidine blue using standard procedures. The sameprocedure in the absence of the primary antibody was performed as anegative control. As a positive control to test the Preservation of thetissue was verified with an antibody specific for the protein Patched1(Santa Cruz Biotechnology). Patched1 was used as positive control sinceit stains all relevant brain structures and is not affected by stress orantidepressants. Very specific staining pattern was detected, consistentwith the specific subcellular localization of P2X7R in brain cells.Negative controls were completely devoid of signal. Positive controlwith Patched1 showed identical signal intensity and distribution in allsamples, indicating that all tissues were equally well preserved andprocessed.

Proceeding from frontal to caudal, P2X7R protein was observed in theglomerular layer of the olfactory bulb at low levels (FIG. 3). P2X7R wasalso present at very low levels in a restricted area of theperiventricular hypothalamic nucleus (FIG. 3). Ependymal cellssurrounding the lateral ventricles also showed a fainted staining (FIG.3). A stronger signal was detected in restricted areas of thehippocampus, where the signal was present in single cells of thepolymorph layer, the lacunosum moleculare and the oriens layer (FIG. 4).In more posterior areas of the hippocampus, the signal was present inthe molecular layer, stratum radiatum and near the CA3. In a furthercaudal position, P2X7R was expressed in the subcomisural organ (FIG. 4).Therefore, the basal P2X7R expression in the brain of stress-free miceis restricted to areas that had been previously associated withdepression, stress, learning and memory.

EXAMPLE 6 P2X7R is Modulated in Mice Treated with an Antidepressant

Further validation of role of P2X7R in affective disorders was performedby examining its expression pattern in response to stress and treatmentwith antidepressant drugs. A treatment schedule which has been proven toproduce antidepressant effects on the behavioural level was administeredto mice which were characterized as antidepressant-responsive by using avariety of behavioural paradigms suitable to detect anxiolytic andantidepressant effects of classical antidepressants like the selectiveserotonin reuptake inhibitor paroxetine. Paroxetine was delivered bygavage to naive male mice over a time period of 28 days at a dosage of10 mg/kg bodyweight twice per day. In parallel, a control group of micewas given vehicle solution (i.e. without paroxetine) using the sametreatment regiment while a second control group of mice was leftundisturbed and stress-free (i.e. untreated) during the same period ofthe experiments. At the end of the long-term treatment, part of the miceof each experimental group were tested in the dark/light box (test ofanxiety behaviour) and in the Porsolt's forced swim test (test ofdepressive-like behaviour) to confirm the effectiveness of the treatment(FIG. 5). Passive stress coping behaviour decreased after long-termtreatment with the antidepressant paroxetine. The other part of theexperimental groups (i.e. mice without test experience) weredecapitated, brains rapidly removed and frozen at −80° C. until usage.

The expression of P2X7R in the brains of mice under stress-freeconditions, and mice under mild stress produced by the vehicleapplication, and mice under paroxetine treatment was evaluated usingthree different brains from each group. Serial slides from each group ofanimals were analyzed in parallel by immunohistochemistry using the samematerials in order to produce completely comparable results. Nosignificant change in P2X7R expression in the olfactory bulb was seen inresponse to stress or to paroxetine treatment (FIG. 6). However, in theperiventricular nucleus of the hypothalamus, paroxetine produced aslight inhibition of P2X7R expression (FIG. 7). No significant changewas observed in the ependymal cells from different brain areas (FIG. 8).The most dramatic changes were observed in the hippocampus, where P2X7Rwas strongly inhibited by stressful handling whereas paroxetinetreatment produced a marked stimulation above basal levels (FIGS. 9, 10and 11). This effect was observed all along the hippocampus but was moreevident in the polymorph layer near the dentate gyrus. In thesubcommissural organ, P2X7R expression remained unchanged by thedifferent treatments. Therefore, P2X7R expression is strongly regulatedin two specific brain areas involved in depression and stress. Otherbrain areas, which showed low levels of P2X7R and are not directlyinvolved in depression, did not show changes.

In the samples from mice treated with paroxetine and showing a strongP2X7R expression, it was possible to analyze the distribution of P2X7Rin more detail (FIGS. 10 and 11). The P2X7R protein was not only presentin cell bodies but also was clearly detected in projections innervatingthe granular layer of the dentate gyrus (FIG. 12). This subcellularlocalization of P2X7R is consistent with a role in neurotransmitterrelease and long term potentiation.

Since some reports (Muria et al., Biochem. J. 288 (1992), 897-901;Ferrari et al., FEBS Lett. 447 (1999), 71-75) suggest that chronic andhigh dose stimulation of P2X7R may cause apoptosis in some cell types,the hippocampus of the above described animals were analyzed for theco-localization of apoptotic cells and P2X7R expressing cells, inconsecutive sections, using TUNNEL staining and immunohistochemistry. Incorrelative sections, only few apoptotic cells were detected and theywere present along the granular layers of the hippocampus where no P2X7Rexpression was observed (FIG. 13). No significant differences in thenumbers of apoptotic cells were observed between the different treatmentconditions. Therefore, the location and number of apoptotic cells didnot correlate with the location and number of cells expressing P2X7R andrules out an involvement of P2X7R in the induction of apoptosis in thehippocampus.

Thus, P2X7R expression is considerably restricted to specific brainareas involved in depression. Moreover, P2X7R expression is inhibited bystress and strongly stimulated by antidepressant treatment in thesespecific areas. Therefore, P2X7R fulfils all criteria required for theactions of antidepressants according to the highest standards in thefield of depression research. In addition, these results suggest thatmodulation of function of P2X7R is associated with chronic stress, whichserves as a model for several aspects of affective disorders.

EXAMPLE 7 The Behavioural Effect of P2X7R Inhibition in Mice

To demonstrate that P2X7R inhibition acts as a causative agent foraffective disorders, P2X7R function was specifically inhibited indistinct regions of the brain without affecting any other brainfunction. This was achieved by delivering double stranded smallinterference RNA molecules (siRNA) into restricted areas of the brain.

According to the observed expression pattern of P2X7R in the hippocampus(FIGS. 9, 10, and 11) and the known involvement of the hippocampus indepression, the dentate gyrus (hippocampus) was selected as targetregion for siRNA application. Male, naive mice were bilaterallyimplanted with a guide cannulae (23 gauge, length 8 mm) by means of astereotactic instrument. The coordinates, in relation to bregma, were−2.0 mm posterior, ±1.0 mm lateral, and −1.0 mm ventral. Following arecovery period of 5 days, the mice were divided into three experimentalgroups: vehicle (veh), control double stranded RNA (control), and P2X7Rspecific double stranded siRNA (siRNA). Sequences used for P2X7R siRNAare 5′-GUGGGUCUUGCACAUGAUCTT-3′ and 5′-GAUCAUGUGCAAGACCCACTT-3′. Bothsequences and were annealed and injected together as a double strandedRNA. On day 6 after surgery, mice were slightly anaesthetized withIsofluran and injections of siRNA were carried out. The concentration ofthe control and siRNA was 0.1 nmol/μl, and a volume of 1 μl per side wasinfused using specifically adapted injection systems (30 gauge, length 9mm). The anaesthesia for the infusion was of short duration and the micewere awake immediately or few seconds after the manipulation.

Once delivered into the brain the siRNA molecules specific for P2X7Rwere taken up by brain cells and specifically induce the degradation ofthe complementary P2X7R mRNA with high efficiency. As a result, P2X7Rfunction was specifically inhibited for a short period without affectingany other brain function. In this regard, injection of vehicle orcontrol siRNA did not result in any obvious changes in normal behaviour,i.e., food and water intake, or motor behaviour in the home cage.

The effects of P2X7R inhibition on depressive-like behaviour wasassessed 24 hours and 48 hours after infusion of siRNA, control orvehicle according to the standard test paradigm, the Porsolt's forcedswim test (Porsolt et al., Arch. Int. Pharmacodym. 229 (1977), 327-336;Porsolt, Rev. Neurosci. 11 (2000), 53-58). The parameter used toevaluate depressive-like behaviour is the time the animal is floating inthe water, a behaviour which is associated with behavioural despair asthe animal does not make any effort to actively cope with the stressfulsituation. Compared to vehicle application, no influence of controldouble stranded RNA (5′-CAACUUCAUCUUCUACGCGTT-3′) on floating behaviour(passive stress coping) was detected. In contrast, compared to controls,mice infused with P2X7R specific siRNA showed a significant increase inpassive behaviour, which is construed as depressive-like behaviour (FIG.14). This interpretation becomes moreover evident when the effects ofantidepressants on passive stress coping behaviour in the forced swimtest are visualized (FIG. 5). Passive stress coping behaviour increasedafter acute intrahippocampal injection (bilateral, dentate gyrus) ofsiRNA targeting P2X7R. The Porsolt's forced swim test is a standard testused to assess the effectiveness of antidepressants and it has beenproven by many studies that the test is selectively sensitive for theseeffects, given that the right animal model is used. The paradigm hasbeen widely used to test pharmaceutical compounds and to validate animalmodels of depression, which show an increase in passive behaviour as dothe mice where P2X7R has been inhibited (siRNA).

At the end of the experiment, the mice were sacrificed and the brainswere examined to confirm the location and efficiency of the siRNAinjections. For this purpose the brains were cut into sections and theslides were stained by immunohistochemistry using the above mentionedprotocols. Brains from mice injected with the specific double strandedsiRNA, with control double stranded RNA and with vehicle were examinedin parallel. Under these conditions, the specific siRNA directed againstP2X7R injected near the dentate gyrus induced on average an 80%inhibition of P2X7R protein expression as compared to the samples frommice injected with vehicle or with control double stranded RNA. Both thenumber of cells expressing P2X7R as well as the intensity of theexpression were strongly reduced (FIG. 15). The injections with siRNAdid not produce any sign of local inflammation or infiltration at thehippocampus. Thus, P2X7R expression is specifically and locallyinhibited by siRNA application in vivo. This inhibition producedbehavioural changes indicating a causative role for P2X7R in affectivedisorders. These results in combination with those mentioned abovesupport and confirm the observation of mutations in P2X7R beingassociated with affective diseases in humans and that modulation ofP2X7R activity has antidepressive effects.

EXAMPLE 8 Drug Screening Assay

Methods for identifying P2X7R agonists were established using animmortalised mouse hippocampal cell line expressing the endogenous P2X7gene. Briefly, the expression of P2X7 was confirmed by culturing thecells at 37° C./5% CO₂ in DMEM with 10% foetal calf serum (Gibco). Uponreaching 80% confluence, cells were collected in PBS and homogenized byrepeated passage through a syringe (18 G needle). The amount of totalprotein was measured by the Bradford assay (Sigma; diluted 1:5, O.D.measured at 595 nm) according to the manufacturer's recommendation.Protein homogenates were then mixed with an equal volume of loadingbuffer (50 mM Tris-Cl pH 6.8; 25% glycerol; 7.2 mM bromophemol blue; 2%SDS; 200 nM β-mercaptoethanol) and subsequently denaturated in boilingwater for 10 minutes. 20 mg of each sample were loaded onto a 10%polyacrylamide gel containing 0.4% SDS. Electrophoresis and Western blottransfer were performed according to conventional protocols described,for example, in Sambrook, Russell “Molecular Cloning, A LaboratoryManual”, Cold Spring Harbor Laboratory, N.Y. (2001). Membranes were thenblocked with 5% dry milk and incubated with an antibody against P2X7R(1:1000 dilution; Santa Cruz Biotech) followed by incubation with ahorse anti-goat peroxidase-coupled secondary antibody (1:10000 dilution;Santa Cruz Biotech). Membranes were then incubated for 1 hour at 37° C.in Lumi-Light Western Blotting Substrate (Roche Applied Science)followed by a 10 minute exposure on a BioMax MR Film (Kodak).

A 70 kD band corresponding to the expected size of the P2X7R protein wasdetected in HT-22 cells demonstrating expression of the endogenous mouseP2X7 gene (FIG. 17). A second mouse hippocampal cell line (HT-39) didnot express P2X7.

Since P2X7R is an ATP-gated ion channel which allows the entry ofcalcium and sodium ions into cells, a method for identifying P2X7Ragonist was established by monitoring calcium influx into HT-22 cells.Cells were first loaded with the fluorescent dye Oregon green AM ester(Molecular Probes) for 30 minutes at room temperature, washed 2 timeswith DMEM/10% foetal calf serum to remove excess dye and cultured for 15minutes in the presence of 100 μM 2- and2′-3′-O-(4-Benzoylbenzoyl)adenosine 5′-triphosphate (BzATP:C₂₄H₂₄N₅O₁₅P₃)). BzATP is a known agonist of P2X7R (North andSurprenant, Annu. Rev. Pharmacol. Toxicol. 40 (2000), 563-580). Calciummovement into the cells was visualised under a fluorescent microscopewith a fluorescein filter (wavelength 492/517 nm). Oregon green AM esteris a fluorescent dye that binds to intracellular calcium. Accordingly,an increase in green fluorescence was observed in cells treated withBzATP (FIG. 18) signalling an activation of P2X7R which results in aninflux of calcium into the cells and an increase binding of Oregon greendye to intracellular calcium.

Alternatively, Oregon green AM ester can be replaced by Fluo-3, fluo-4,fluo-5F, fluo-5N, fluo-4FF, Fluo-4 dextran, Fluo-3 AM, Fluo-4 AM,Fluo-5F AM, Fluo-5N AM and Fluo-4FF AM (Molecular Probes). Calciuminflux in HT-22 cells can also be measured in 96-well and 384-wellmicroplate using the Calcium Plus Assay Kit (Molecular Device) or FLIPR®Calcium Assay Kit for Fluorometric Imaging Plate Reader Systems(Molecular Device). HT-22 cells can be replaced by any cells expressingP2X7R, including cells that have been genetically modified byintroducing an exogenous P2X7 gene.

The specificity of the P2X7R agonist on calcium influx was confirmed bypre-treatment of HT-22 cells with 100 mM Oxidized ATP (oATP; Sigma) for1 hour before the addition of BzATP. oATP is an irreversible inhibitorof the receptor (Chen et al., J. Biol. Chem., 268 (1993), 8199-8203).Activation of P2X7R by the agonist was inhibited by oATP (FIG. 18) asillustrated by the absence of green fluorescence in the cells.

Yet another method of measuring P2X7R activity involves the entry ofethidium bromide into P2X7R expressing cells. Activation of P2X7R by anagonist allows the entry of ethidium bromide which binds nuclear DNA andemits a fluorescence signal. Alternatively, the propidium dye YOPRO-1can be substituted for ethidium bromide. An increase in fluorescence canbe used as a measure of P2X7 receptor activation. Therefore, the assaycan be used to test and quantify the effect of an agent or compound withagonist properties on P2X7R. In the present example, 10³ HT-22 cellswere seeded per well in a 96-well flat bottom microtitre plates andincubated at 37° C./5% CO₂ in DMEM medium containing 10% FCS until thecells attached to the culture surface. Once attached, cells wereincubated for 60 minutes in DMEM medium containing 10% FCS, 10⁻⁴Methidium bromide and increasing concentrations of BzATP (1 μM, 10 μM,100 μM, 500 μM, 1 mM). The number of fluorescent cells which haveintegrated the ethidium bromide to the DNA can then be counted using afluorescent microscope (Zeiss, Germany). Concentrations above 100 μMBzATP increased the number of fluorescent nuclei signalling activationof P2X7R (FIG. 19 a). Alternatively, ethidium bromide fluorescence canbe measured using a Perkin-Elmer fluorescent plate reader (excitation520 nm, emission 595 nm, slit widths: Ex 15 nm, Em 20 nm). From thereadings obtained, a pIC50 figure can be calculated for each candidateagent or compound. Accordingly, a P2X7R agonist is defined as an agentor a compound with an EC50 equal or below 300 micromolar, whereas theterm EC 50 is defined as the concentration eliciting 50% of maximalresponse to an agonist (North and Surprenant, Annu. Rev. Pharmacol.Toxicol. 40 (2000), 563-580). The specificity of an agonist for P2X7Rcan be evaluated by pre-incubation of the cells for 60 minutes with 100μM o-ATP before adding the agonist and ethidium bromide dye. Under theseconditions, activation of P2X7R by the agonist is inhibited by oATPresulting in a reduction in the number of fluorescent cells (FIG. 19 b).

Yet another method for identifying P2X7R agonists was devised bygenerating a immortalised mouse cell line that overexpresses the humanP2X7R gene under the control of the human cytomegalovirus (CMV) earlypromoter/enhancer region. The human P2X7R cDNA was inserted into thepcDNA3.1 vector (Invitrogen) and transfected into the mouse hippocampalcell line HT-22 using Lipofectamine (Invitrogen) according to themanufacturer's specifications. One day after transfection, culturemedium containing 500 μg/ml G418 was added to the cells. Resistantclones were separately isolated and cultured 14 days after applying theselection medium.

The agonistic activity of a compound was evaluated by measuring calciumentry in the cells that overexpress the human P2X7R. Cells were culturedin 96 well plates and incubated at 37° C. with 5% CO₂ DMEM with 10%foetal calf serum (Gibco) until they reached confluence. Cells were thenloaded for one hour with 10 μM of Fluo-4 AM (Molecular Probes). Fluo-4AM is a fluorescent dye that binds to intracellular calcium. Afterloading, cells were washed once with a buffer containing 0.5 mM CaCl₂and 20 mM Hepes and were treated with 20 μM BzATP or 50 μM tenidap.Agonist activity was detected by measuring an increase in calcium influxwhich results in increased binding to Fluo-4 AM and increasedfluorescence. Changes in fluorescence signal are measured using aFluostar Optima plate reader (BMG biotech). Both BzATP and tenidapproduced a rapid increase in fluorescence intensity which declinedslowly over time (FIG. 19 c). Thus, both compound stimulated theactivity of P2X7R which results in an influx of ions into the cells.

EXAMPLE 9 Activation of P2X7R with Agonists has Antidepressive Effects

To demonstrate that activation of P2X7R has therapeutic effects onaffective disorders, the P2X7R agonist BzATP(2′-3′-O-(4-Benzoylbenzoyl)adenosine 5′-triphosphate (C₂₄H₂₄N₅O₁₅P₃))was administered to a selected DBA/201a mouse strain that displayscharacteristics of being highly anxious, responding to antidepressants,and showing anxiolysis after subchronic antidepressant treatment (Luckiet al., Psychopharmacology 155 (2001), 315-322). BzATP is a compoundwith strong specificity to P2X7R (North and Surprenant, Annu. Rev.Pharmacol. Toxicol. 40 (2000), 563-580). In the present example, theP2X7R agonist was directly injected into the hippocampus of mice.However, a P2X7R agonist agent or compound could also be deliveredorally, subcutaneously, intravenously, intra-arterial, intranodal,intramedullary, intrathecal, intraventricular, intranasally,intrabronchial, transdermally, intrarectally, intraperitoneally,intramuscularly, intrapulmonary, vaginally, rectally, or intraocularly.

Four months old male mice were bilaterally implanted with guide cannulae(23 gauge, length 8 mm) by means of a stereotactic instrument (DavidKopf Instruments). The coordinates, in relation to bregma, were −2.0 mmposterior, ±1.0 mm lateral, and −1.0 mm ventral. After surgery, the micewere allowed to recover for 10 to 12 days. Following this recoveryperiod, mice were injected with 1 μl vehicle solution (0.5% DMSO, Sigma)or 50 μM BzATP (Sigma, prepared in 0.5% DMSO) in each side of the brainover a period of 60 seconds. Injections were performed using a 9 mm-30gauge needle inserted into the guide cannulae and connected via tubingto a 10 μl Hamilton syringe.

The behaviour of individual mice was assessed using the Porsolt's forcedswim test 24 hours after injection of vehicle solution or BzATP. Apre-exposure of 5 minutes to the test was done 10-15 minutes aftervehicle or BzATP injection. The forced swim test is a standard test thatmeasures primary stress-induced reductions in avoidance or escape,termed behavioural despair. The test is used to determine theeffectiveness of antidepressants, test new pharmaceutical compounds andvalidate animal models of depression (Porsolt et al., Arch. Int.Pharmacodym. 229 (1977), 327-336; Porsolt, Rev. Neurosci. 11 (2000),53-58; Rénéric et al., Behav. Brain Res. 136 (2002), 521-532; Page etal., Psychopharmacology 165 (2003), 194-201; Kelliher et al.,Psychoneuroendocrinology 28 (2003), 332-347). The test consists ofplacing a mouse for a period of 5 minutes into a glass cylindercontaining water. Under such circumstances, the mouse cannot touch thebottom of the cylinder and is thus forced to swim. Time, latency andfrequency of struggling versus floating are scored as behaviouralparameters. Floating (i.e. movements made only for keeping balance andbreath) is a passive behaviour associated with despair and represents adepressive-like symptom since the animal does not make any effort toactively cope with the stressful situation. Increased struggling (i.e.active attempts to escape) indicates active coping behaviour that can beinterpreted as an improvement of depression-like symptoms. For example,treatment with serotonergic antidepressants reduce the total time spentfloating (Borsini, Neurosci. Biobehav. Rev. 19 (1995), 377-395; Redrobeand Bourin, Psychopharmacology 138 (1998), 198-206, and in parallelincreases the time of active behaviour (i.e. swimming or struggling;Lucki et al., Psychopharmacology 155 (2001), 315-322).

The P2X7R agonist BzATP was found to increase active escape attempts(i.e. increase in time and frequency of struggling, decrease in latencyof struggling) while a decrease in passive behaviour (i.e. decrease intime and frequency of floating, increase in latency of floating) wasmeasured when compared to control mice injected with vehicle solution(FIG. 20). Observed results were verified statistically usingMann-Whitney U and one-way MANOVA tests. The differences in timestruggling, latency of floating and frequency of floating were found tobe statistically significant. While latency and frequency of strugglingand time floating results were not supported statistically, they stillrepresented a tendency towards improvement in stress coping behaviour.These results demonstrate that a P2X7R agonist can lead to improvementsin depressive-like symptoms.

Since conclusions drawn from the forced swim test can be influenced byunspecific effects of an agent or compound on animal activity (i.e.increase in struggling behaviour can be the result of hyperactivityinstead of increased active coping behaviour), the potential effect ofBzATP on locomotor activity was assessed by the open field test (Crawley“What's wrong with my mouse: Behavioral phenotyping of transgenic andknockout mice”, Wiley-Liss (2000)). Locomotor activity in mice treatedwith control vehicle solution or 50 μM BzATP was assessed 24 hours afterinjection by placing individual animal in a dark-grey wooden box(30×30×40 cm). Locomotor activity was monitored for a period of 30minutes using a video camera. Overall distance travelled by the animalsduring the testing period was then analysed by means of VideoMot2computer software (TSE GmbH, Bad Homburg). No difference in locomotoractivity was measured between mice treated with control vehicle solutionand BzATP (FIG. 21). Therefore, the application of BzATP did not inducehyperactivity. These results confirm that activation of P2X7R by anagonist agent or compound leads to improvements in depressive-likesymptoms and is not the result of an unspecific effect on animalactivity per se.

Several reports suggest that activation of P2X7R can induce apoptosisand cell death in vitro (Di Virgilio et al., Cell Death Differ. 5(1998), 191-199, Virginio et al., J. Physiol. 519 (1999), 335-346). Totest whether P2X7R activation in the hippocampus resulted in cell death,apoptosis levels were quantified in the brain of the mice treated withBzATP. Mice were sacrificed at the end of the behavioural experiments,the brains were removed, shock frozen and sectioned into 16 μm slices.Brain sections were then studied for apoptosis using the DeadEndfluorometric TUNEL system according to the manufacturer's recommendation(Promega Corporation). The TUNEL system measures the fragmented DNA ofapoptotic cells. Positive control for the assay are made by pre-treatingbrain sections for 10 minutes with 1 unit/ml of DNAse I.

Very few apoptotic cells (i.e. less than one cell per brain section)were observed in brains of mice treated with control vehicle or theP2X7R agonist (FIG. 22) when compared to positive control sectionspre-treated with DNAse. Moreover, no significant differences in thenumbers of apoptotic cells was observed between the control animals andmice treated with BzATP, indicating that activation of P2X7R did notresult in cerebral cell death in vivo.

EXAMPLE 10 P2X7R Antagonists Have no Antidepressive Effects

The P2X7R antagonists KN-62(1-(N,O-bis[5-isoquinolinesulphonyl]-N-methyl-L-tyrosyl)-4-phenylpiperazine)and oxidized ATP (oATP) were administered to DBA/201a mice (HarlanWinkelmann, Germany) that exhibit the behavioural characteristic ofbeing highly anxious. KN-62 has been shown to be a non competitiveantagonist of P2X7R (Chessel et al., Brit. J. Pharmacol., 124 (1998),1314-1320) while oATP acts as an irreversible inhibitor of P2X7R (Chenet al., J. Biol. Chem., 268 (1993), 8199-8203).

In the present example, the P2X7R antagonists were directly injectedinto the dentate gyrus region of the hippocampus. Briefly, three monthsold male mice were bilaterally implanted with guide cannulae (23 gauge,length 8 mm) by means of a stereotactic instrument (David KopfInstruments). The coordinates, in relation to bregma, were −1.5 mmposterior, ±1.0 mm lateral, and −0.8 mm ventral. Mice were allowed torecover for 10 to 13 days after surgery. Following this recovery period,mice were injected with 1 μl vehicle solution (0.01% DMSO, Sigma), or100 nM KN-62 (Sigma, prepared in 0.01% DMSO), or 10 μM oATP (Sigma,prepared in PBS) in each side of the brain over a period of 60 seconds.All injections were performed using a 9 mm-31 gauge needle inserted intothe guide cannulae and connected via tubing to a 10 μl Hamilton syringe.

The behaviour of individual mice was assessed using the Porsolt's forcedswim test 24 hours after injection of vehicle solution, KN-62, or oATP.A pre-exposure of 5 minutes to the test was performed 15-17 minutesafter administration of vehicle, KN-62, or oATP. A description of thePorsolt's forced swim test is given in example 9. In the presentexample, no changes in active escape attempts (i.e. time, frequency,latency of struggling) or in passive behaviour (i.e. time, frequency,latency of floating) was measured between vehicle, KN-62 or oATP treatedmice (FIG. 23). Observed results were verified statistically usingone-way MANOVA test. The differences seen in the different parametersbetween vehicle, KN-62 or oATP treated mice were not supportedstatistically. These results demonstrate that P2X7R antagonists do notimprove depressive-like symptoms and have no antidepressive action.

Since conclusions drawn from the forced swim test can be influenced byunspecific effects of an agent or compound on animal activity (i.e.increase in struggling behaviour can be the result of hyperactivityinstead of increased active coping behaviour), the potential effect ofthe P2X7R antagonist oATP on locomotor activity was assessed byperforming the open field test. Locomotor activity in mice treated withcontrol vehicle solution, 10 μM oATP, or 50 μM oATP was assessed 15minutes after injection by placing individual animal in a dark-greywooden box (30×30×40 cm). Locomotor activity was monitored for a periodof 30 minutes using a video camera. Overall distance travelled by theanimals during the testing period was then analysed by means ofVideoMot2 computer software (TSE GmbH, Bad Homburg). No difference inlocomotor activity was measured between mice treated with controlvehicle solution and oATP (FIG. 24). Therefore, the application of aP2X7R antagonist did not induce hypo- or hyperactivity in the animals.

1. An isolated nucleic acid molecule comprising a nucleic acid sequenceselected from the group consisting of: (a) a genomic nucleotide sequenceencoding an ATP-gated ion channel P2X7R and which contains a mutation inthe 5′UTR region corresponding to positions 362, 532, 1100, 1122, 1171or 1702 of the genomic sequence of the wild-type ATP-gated ion channelP2X7R as depicted in SEQ ID NO: 1, wherein at said position saidnucleotide is replaced by another nucleotide; (b) a nucleic acidsequence encoding a polypeptide which has an amino acid sequence of theATP-gated ion channel P2X7R, wherein in the exon as indicated in column“Exon” of the following Table A the amino acid residue as indicated incolumn “Amino acid residue” of Table A corresponding to the position asindicated in column “Position in wild-type” of Table A of the wild-typeATP-gated ion channel P2X7R amino acid sequence as depicted in SEQ IDNO: 3 or 4 is replaced by another amino acid residue TABLE A Exon Aminoacid residue Position in wild-type exon 3 R (Arg) 117 exon 5 G (Gly) 150exon 6 E (Glu) 186 exon 6 L (Leu) 191 exon 8 R (Arg) 270 exon 13 I (Ile)568 exon 13 R (Arg) 578

(c) a nucleotide sequence encoding an ATP-gated ion channel P2X7R andwhich contains a mutation in exon 5 or 8 corresponding to position 32548or position 37633 of the wild-type ATP-gated ion channel P2X7Rnucleotide sequence as depicted in SEQ ID NO: 1, wherein at saidposition said nucleotide is replaced by another nucleotide (d) a nucleicacid sequence encoding a polypeptide which has an amino acid sequence ofan ATP-gated ion channel P2X7R, wherein amino acids corresponding topositions 488 to 494 of the wild-type ATP-gated ion channel P2X7R asdepicted in SEQ ID NO: 3 or 4 are deleted; (e) a genomic nucleotidesequence encoding an ATP-gated ion channel P2X7R, wherein in the intronas indicated in column “Intron” of the following Table B the nucleotideas indicated in column “Replaced nucleotide” of Table B corresponding tothe position as indicated in column “Position in wild-type” of Table Bof the wild-type ATP-gated ion channel P2X7R nucleotide sequence asdepicted in SEQ ID NO: 1 is replaced by another nucleotide TABLE BREPLACED Intron NUCLEOTIDE Position in wild-type intron 1 G 3166 intron1 C 24778 intron 1 C 24830 intron 3 A 26308 intron 3 G 26422 intron 4 G32394 intron 4 T 32434 intron 5 A 32783 intron 6 G 35641 intron 6 A35725 intron 6 T 36001 intron 7 G 36378 intron 7 T 36387 intron 7 G36398 intron 9 C 47214 intron 11 T 47563 intron 12 C 54307 intron 12 G54308

(f) a genomic nucleotide sequence encoding an ATP-gated ion channelP2X7R and which contains a mutation in the 3′UTR region corresponding toposition 54925, 55169, 55170, 55171 or 55917 of the wild-type ATP-gatedion channel P2X7R nucleotide sequence as depicted in SEQ ID NO: 1,wherein at said position said nucleotide is replaced by anothernucleotide; (g) a nucleotide sequence comprising at least 20 or 21nucleotides and comprising the mutations or deletions as defined in anyone of (a) to (f); (h) a nucleic acid sequence comprising a nucleotidesequence as shown in any one of SEQ ID NOs: 13 to 51; (i) a nucleic acidsequence encoding a polypeptide comprising the amino acid sequence ofSEQ ID NOs: 5 to 12; (j) a nucleotide sequence which hybridizes to anucleotide sequence defined in any one of (a) to (g) or to thenucleotide sequence of (h) and having a mutation as defined in any oneof (a) to (f); and (k) a nucleic acid sequence being degenerate as aresult of the genetic code to the nucleic acid sequence as defined in(j).
 2. The nucleic acid molecule of claim 1 derived from mouse, rat orhuman.
 3. The nucleic acid molecule of claim 1 which is DNA, RNA, PNA orphosphorothioates.
 4. A vector comprising the nucleic acid molecule ofclaim
 1. 5. The vector of claim 4 which is an expression vector, a genetargeting vector and/or a gene transfer vector.
 6. A host transformedwith the nucleic acid molecule of claim 1 or a vector comprising saidnucleic acid molecule.
 7. The host of claim 6 which is a mammalian cell,an amphibian cell, a fish, an insect cell, a fungal cell, a plant cellor a bacterial cell.
 8. The host of claim 7, wherein said mammalian cellis selected from the group consisting of CHO cells, HEK293 cells, COS-7cells or PC12 cells.
 9. The host of claim 7, wherein said amphibian cellis an oocyte, preferably a Xenopus oocyte.
 10. The host of claim 9,wherein said oocyte is a frog oocyte.
 11. The host of claim 6 which is anon-human transgenic organism.
 12. The host of claim 11, wherein saidnon-human organism is a mammal, amphibian, a fish, an insect, a fungusor a plant.
 13. A method for producing the polypeptide encoded by anucleic acid molecule of claim 1(b) or 1(d) comprising culturing/raisinga host transformed with the nucleic acid molecule of claim 1 or a vectorcomprising said nucleic acid molecule and isolating the producedpolypeptide.
 14. A polypeptide encoded by the nucleic acid molecule ofclaim 1(b) or 1(d) or produced by a method comprising culturing/raisinga host transformed with the nucleic acid molecule of claim 1 or a vectorcomprising said nucleic acid molecule.
 15. An antibody specificallydirected to the polypeptide of claim 14, wherein said antibodyspecifically reacts with an epitope generated and/or formed by themutation in the ATP-gated ion channel P2X7R selected from the groupconsisting of: (i) an epitope specifically presented by a polypeptidewhich has an amino acid sequence of an ATP-gated ion channel P2X7R,wherein the R (Arg), G (Gly), E (Glu), L (Leu), R (Arg), I (Ile) or R(Arg) residue corresponding to position 117, 150, 186, 191, 270, 568 or578 of the wild-type ATP-gated ion channel P2X7R as depicted in SEQ IDNO: 3 or 4 is replaced by another amino acid residue; and (ii) anepitope specifically presented by a polypeptide which has an amino acidsequence of an ATP-gated ion channel P2X7R, wherein amino acidscorresponding to positions 488 to 494 of the wild-type ATP-gated ionchannel P2X7R as depicted in SEQ ID NO: 3 or 4 are deleted.
 16. Theantibody of claim 15 which is a monoclonal antibody.
 17. An aptamerspecifically binding to a nucleic acid molecule of claim 1 or to thepolypeptide encoded by the nucleic acid molecule of claim
 1. 18. Aprimer or pair of primers capable of specifically amplifying a nucleicacid molecule as defined in claim
 1. 19. The primer or pair of primersof claim 18, which is selected from the group consisting of SEQ ID NOs.:52 to
 111. 20. A composition comprising the nucleic acid molecule ofclaim 1, a vector comprising said nucleic acid molecule, a polypeptideencoded by said nucleic acid molecule, an antibody specifically directedto said polypeptide, an aptamer specifically binding to said nucleicacid molecule and/or a primer or pair of primers capable of specificallyamplifying said nucleic acid molecule.
 21. The composition of claim 20which is a diagnostic composition.
 22. The composition of claim 21,optionally further comprising suitable means for detection. 23.(canceled)
 24. A method of diagnosing an affective disorder or asusceptibility to an affective disorder comprising the step ofdetermining in a sample obtained from an individual whether the P2XR7protein expressed in the cells of said individual is non-functional,shows an altered ATP-gating in comparison to the wild-type P2XR7 proteinor is over- or under-expressed in comparison to the P2XR7 protein levelan unaffected individual.
 25. A method for diagnosing an affectivedisorder or a susceptibility to an affective disorder comprising thestep of determining in a sample obtained from an individual whether theP2X7R gene sequence or encoded protein thereof comprises a mutation incomparison to the wild-type P2X7R sequence.
 26. A method for diagnosingan affective disorder or a susceptibility to an affective disordercomprising the step of determining in a sample obtained from anindividual whether the P2X7R gene sequence or encoded protein thereofcomprises a mutation in comparison to the wild-type P2X7R sequence,wherein said mutation is a mutation as defined in claim 1 and/or anucleotide replacement or deletion selected from the following Table Cindicating in column “Region of P2X7R” the region of the P2X7R genomicnucleotide sequence in which the replacement or deletion occurs, incolumn “Nucleotide” of Table C the nucleotide which is replaced byanother nucleotide or the nucleotides which are deleted and in column“Position in wild-type” of Table C the corresponding position in thenucleotide sequence of the wild-type ATP-gated ion channel P2X7R asdepicted in SEQ ID NO: 1 TABLE C Region of Position in P2X7R NUCLEOTIDEwild-type 5′UTR T 362 5′UTR T 532 5′UTR A 1100 5′UTR A 1122 5′UTR C 11715′UTR T 1351 5′UTR G 1702 5′UTR T 1731 5′UTR C 1860 5′UTR C 2162 5′UTR C2238 5′UTR A 2373 5′UTR G 2569 5′UTR G 2702 intron 1 G 3166 intron 1 C24778 intron 1 C 24830 exon 2 T 24942 exon 3 C 26188 exon 3 A 26308 exon3 G 26422 intron 4 G 32394 intron 4 T 32434 exon 5 G 32493 exon 5 G32506 exon 5 C 32507 exon 5 C 32548 intron 5 A 32783 intron 5 T 35309intron 5 C 35374 intron 5 A 35378 exon 6 G 35438 exon 6 T 35454 intron 6T 35549 intron 6 G 35641 intron 6 A 35725 intron 6 T 36001 intron 6 A36064 intron 6 deletion of GTTT 36091 to 36094 intron 6 C 36108 intron 7C 36374 intron 7 G 36378 intron 7 T 36387 intron 7 G 36398 intron 7 C37439 intron 7 T 37513 exon 8 C 37604 exon 8 G 37605 exon 8 G 37623 exon8 C 37633 intron 9 C 47214 exon 11 G 47383 exon 11 C 47411 intron 11 T47563 intron 12 C 54307 intron 12 G 54308 exon 13 C 54399 exon 13 A54480 exon 13 C 54523 exon 13 deletion of 54562 to CCCTGAGAGCCACAGGTGCCT54582 exon 13 A 54588 exon 13 C 54664 exon 13 G 54703 exon 13 A 54804exon 13 G 54834 exon 13 G 54847 3′UTR G 54925 3′UTR C 55169 3′UTR A55170 3′UTR A 55171 3′UTR C 55917


27. The method of claim 26, wherein the occurrence of the mutation inthe ATP-gated ion channel P2X7R gene is determined by PCR orimmunological methods.
 28. The composition of claim 20 which is apharmaceutical composition.
 29. The pharmaceutical composition of claim28, optionally further comprising a pharmaceutically acceptable carrier.30. A method of treating an affective disorder comprising administeringa therapeutically effective amount of the nucleic acid molecule asdefined in claim 1 or a therapeutically effective amount of apolypeptide encoded by said nucleic acid molecule to a subject sufferingfrom said disorder.
 31. (canceled)
 32. A pharmaceutical compositioncomprising a nucleic acid molecule comprising a nucleotide sequencewhich encodes a functional ATP-gated ion channel P2X7R and which isselected from the group consisting of: (a) a nucleotide sequenceencoding a polypeptide comprising the amino acid sequence as depicted inSEQ ID NO: 3 or 4; (b) a nucleotide sequence comprising the nucleotidesequence as depicted in SEQ ID NO: 1 or 2; (c) a nucleotide sequencewhich hybridizes to the nucleotide sequence of (a) or (b); and (d) anucleotide sequence which is degenerated as a result of the genetic codeto the nucleotide sequence of (c).
 33. A pharmaceutical compositioncomprising a compound the administration of which to cells leads to anincrease of the expression of a nucleic acid encoding an ATP-gated ionchannel P2X7R in the cells or comprising a nucleic acid molecule theexpression of which in cells or the administration of which to cellsleads to an increase of the expression of a nucleic acid encoding anATP-gated ion channel P2X7R in the cells.
 34. A method of treating anaffective disorder comprising administering a therapeutically effectiveamount of the pharmaceutical composition as defined in claim 32, apharmaceutical composition comprising a compound the administration ofwhich to cells leads to an increase of the expression of a nucleic acidencoding an ATP-gated ion channel P2X7R in the cells, a pharmaceuticalcomposition comprising a nucleic acid molecule the expression of whichin cells or the administration of which to cells leads to an increase ofthe expression of a nucleic acid encoding an ATP-gated ion channel P2X7Rin the cells or a pharmaceutical composition comprising a polypeptideencoded by said nucleic acid molecule thereof to a subject sufferingfrom said disorder.
 35. (canceled)
 36. A method of treating an affectivedisorder comprising administering a therapeutically effective amount ofa pharmaceutical composition comprising a modulator of P2X7R activity toa subject suffering from said affective disorder.
 37. The method ofclaim 36, wherein said modulator is an agonist.
 38. The method of claim37, wherein said agonist is selected from the group consisting of ATP,ATP-4 and BzATP (2′-3′-O-(4-Benzoylbenzoyl)adenosine 5′-triphosphate(C₂₄H₂₄N₅O₁₅P₃)).
 39. The method of claim 37, wherein said agonist istenidap (C₁₅H₁₁ClN₂O₂S) or a derivative thereof or3-substituted-2-oxindole-1-carboxamides.
 40. The method of claim 36wherein said pharmaceutical composition optionally further comprises aβ-adrenergic receptor modulator.
 41. The method of claim 40, whereinsaid β-adrenergic receptor modulator is a β-adrenergic receptorantagonist selected from the group consisting of DL-propanolol,D-propanolol and labetolol.
 42. The method of claim 36, wherein saidaffective disorder is selected from the group consisting of majordepression, generalized anxiety disorder and bipolar disorder.
 43. Themethod of claim 42, wherein said major depression is selected from thegroup consisting of major depression, dysthymia, atypical depression,premenstrual dysphoric disorder and seasonal affective disorder.
 44. Themethod of claim 42, wherein said generalized anxiety disorder isselected from the group consisting of panic disorder, phobias,agoraphobia, social phobia, specific phobia, obsessive-compulsivedisorder, post-traumatic stress disorder, separation anxiety disorder,mania, hypomania and cyclothymic disorder.
 45. The method of claim 42,wherein said bipolar disorder is bipolar disorder type I or bipolardisorder type II.
 46. A kit comprising the nucleic acid molecule ofclaim 1, a vector comprising said nucleic acid molecule, a hostcomprising said nucleic acid molecule, a polypeptide encoded by saidnucleic acid molecule, an antibody specifically directed to saidpolypeptide, an aptamer specifically binding to said nucleic acidmolecule and/or a primer or pair of primers capable of specificallyamplifying said nucleic acid molecule.
 47. A method for identifyingcompounds which are capable of specifically interacting with thepolypeptide of claim 14, comprising the steps of contacting apolypeptide of claim 14 with a compound or a candidate mixture ofcompounds to be tested; and determining whether said compound or acandidate mixture of compounds is capable of specifically interactingwith said polypeptide.
 48. A method for the characterization ofcompounds which are capable of altering characteristics of thepolypeptide of claim 14, comprising the steps of contacting apolypeptide of claim 14 with a compound or a candidate mixture; anddetermining whether the compound or a candidate mixture alters acharacteristic of the polypeptide of claim
 14. 49. A method of screeningfor compounds which are capable of interacting with the polypeptide ofclaim 14, comprising the steps of (a) contacting a polypeptide of claim14 with a compound or a candidate mixture of compounds; (b) measuringand/or detecting a response; and (c) comparing said response to astandard response as measured in the absence of said candidate molecule.50. A method for the production of a pharmaceutical compositioncomprising the steps of the method of claim 47 and comprising a furtherstep, wherein a derivative of said identified, characterized and/orscreened molecule is generated.
 51. A method for the production of apharmaceutical composition comprising the steps of the method of claim47 and formulating the molecules identified, characterized, screenedand/or derivatized in pharmaceutically acceptable form.
 52. The methodof claim 51, wherein the pharmaceutical composition to be producedfurther comprises neuroprotective substances, nootrophic substances,brilliant blue, piperidine or piperazine derivatives thereof, adamantinederivatives, substituted phenyl compounds, oxidized ATP,2-O-(4-benzoylbenzoyl)adenosine-5-triphosphate,3-O-(4-benzoylbenzoyl)adenosine-5-triphosphate or β-adrenergic receptormodulators.
 53. The method of claim 47, wherein said compound(s) orcandidate mixture(s) of compounds comprise(s) antagonist(s), partialantagonist(s), partial agonist(s) and/or agonist(s) for an alteredATP-gated ion channel P2X7R.
 54. A method for diagnosing an affectivedisorder of an individual comprising: (a) isolating DNA from cellsobtained from an individual; (b) determining all or part of thenucleotide composition of the P2X7R gene; and (c) analyzing saidnucleotide composition of P2X7R for the presence of one or morepolymorphism(s) mutation or allelic variation.
 55. A method fordiagnosing an affective disorder of an individual comprising: (a)isolating RNA from cells obtained from an individual; (b) convertingsaid RNA into cDNA; (c) determining all or part of the nucleotidecomposition of the P2X7R gene; and (d) analyzing said nucleotidecomposition of P2X7R for the presence of one or more polymorphism(s),mutation or allelic variation.
 56. A method for diagnosing an affectivedisorder of an individual comprising: (a) isolating RNA or protein fromcells obtained from an individual; (b) determining the levels of P2X7RRNA or protein; and (c) comparing the levels of P2X7R RNA or proteinwith the corresponding levels from a normal individual not afflictedwith an affective disorder.